ES2613662T3 - Multifunctional alleles - Google Patents

Abstract

A nucleic acid construct, comprising: (a) target fixing arms to direct the nucleic acid construct to a target gene of a nucleic acid of a cell; (b) a sense-oriented drive sequence with respect to transcription of the target gene, and a drug selection cassette (DSC) in sense or antisense orientation; (c) in antisense orientation, a nucleotide sequence of interest (NSI) and a COIN (conditional element by inversion); and (d) recombinant units, wherein the recombinant units comprise two first pairs of cogninate recombinase recognition sites recognized by a first recombinase, a first recombinant unit comprising the driving sequence, the DSC and the NSI, wherein the first unit Recombinant is flanked by a pair of the first two pairs of cognin recombinase recognition sites recognized by the first recombinase, a second recombinant unit comprising the NSI, wherein the second recombinant unit is flanked by a pair of the first two pairs of sites of recombinase recognition cognatos recognized by the first recombinase, two second pairs of recombinase recognition sites cognatos recognized by a second recombinase, and a third pair of recognition sites of recombinase cognatos recognized by a third recombinase; wherein the first pairs, the second pairs and the third pairs of recombinase recognition sites are recognized by different recombinases; wherein the recombinant units recombine after exposure to a first recombinase to form a conditional allele that lacks the drive sequence and the DSC and contains the NSI in sense orientation and the COIN in antisense orientation; and wherein the conditional allele, when additionally exposed to the second recombinase, recombines to form an allele that lacks NSI and has the COIN in a sense orientation.

Description

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DESCRIPTION

Multifunctional alleles FIELD OF THE INVENTION

The invention relates to nucleic acid constructs for genome modification, including inactivated constructs and constructs for placing COINs in a genome. Genetically modified non-human animals are described, e.g. eg, genetically modified mice that have nucleic acid genes or elements arranged with selected recombinase recognition sites that allow deletion or inversion of the nucleic acid genes or elements to form null alleles, selectable alleles, reporter alleles and / or conditional alleles in non-human animals, p. eg, in mice and rats.

BACKGROUND

Typically, inactivations are performed by homologous substitution of a target gene with another sequence of choice, usually a reporter cassette and a selection cassette, in which the latter is preferably flanked by site-specific recombinase sites to strengthen the separation of the target. Selection cassette through the action of the site-specific cognate recombinase. The selection cassette can be subsequently separated, either by treatment of the cells with the corresponding cognate recombinase or by the progeny of mice to a "suppressor" strain. For example, in the case of "floxed" alleles (in which the sequence of interest is flanked by loxP sites), the cognate recombinase is Cre and what remains in the genome is a single loxP site and the reporter.

Traditionally a related strategy has been used to generate null conditional alleles. This involves flanking part of the gene of interest with site specific recombinase recognition sites (such as lox for Cre and FRT for Flp) in such a way that after the action of the cognate recombinase, the region flanked by the recognition sites of Site specific recombinase are removed and the resulting allele is a null allele.

Although attempts have been made to incorporate both null and conditional functionality in a target fixing vector and to carry out the construction of the corresponding modified alleles in a single target setting stage, the methods that have resulted from such attempts have several inconveniences and have had a contradictory success. These drawbacks include, for example, the lack of true functionality (that is, the null version is not a true null, the conditional allele is not a true conditional, the lack of function of the informer, etc.) or the inability to obtain an allele of practical work with the desired characteristics.

Therefore, there is a need in the art for the generation of genetically modified organisms through the objective setting, in which the loci modified by genetic engineering are multifunctional loci, for example, a first true KO allele and then a null allele conditional or other conditional mutant allele.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the use of recombinase recognition sites to simultaneously remove one element (U) and reverse another (D).

FIG. 2 illustrates an embodiment of a Multifunctional Allele (MFA), shown for a nucleotide sequence of interest (NSI), the use of a splice acceptor with the splice region and an actuation sequence, followed by a signal polyadenylation (pA), a drug selection cassette (DSC; in an appropriate orientation of choice), a COIN and five pairs of recombinase recognition sites. R1 / R1 ', R2 / R2', R3 / R3 ', R4 / R4' and R5 / R5 'represent cognitive pairs of recombinase recognition sites.

FIG. 3 illustrates a conceptual representation of recombinant units (defined by R1 / R1 ', R2 / R2', R3 / R3 ', R4 / R4' and R5 / R5 ') of an embodiment of an MFA allele employing an actuation sequence ( for simplicity, the splice acceptor and the splice region preceding the sequence, and the polyA signal following the sequence are not shown), a DSC (in an appropriate orientation of choice), an NSI and a COIN

FIG. 4 illustrates a particular embodiment of an MFA with specific recombinase recognition sites for purposes of illustration (upper part) and which generates a "cleaned" null allele comprising a drive sequence comprising a LacZ sequence, and extraction of the DSC, as well as the NSI and the COIN element of the initial embodiment of the MFA using a single recombinase stage. For simplicity, the splice acceptor and the splice region preceding the LacZ sequence, and the polyA signal following the LacZ sequence are not shown.

FIG. 5 illustrates a particular embodiment of an MFA that generates a conditional allele that contains / incorporates a COIN of an MFA using a single recombinase (here, a Flp recombinase that first acts on FRT3 sites of the allele embodiment). For simplicity, the splice acceptor and the splice region preceding the LacZ sequence, and the polyA signal following the LacZ sequence are not shown.

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FIG. 6 illustrates an embodiment of an MFA that generates a conditional null allele that contains / incorporates a COIN of an MFA using a single recombinase (aqrn, a Flp recombinase that first acts on FRT sites of the allele embodiment). For simplicity, the splice acceptor and the splice region preceding the LacZ sequence, and the polyA signal following the LacZ sequence are not shown.

FIG. 7 illustrates an embodiment where, after treatment with recombinase (Flp exposure as shown in FIG. 5 or FIG. 6), the allele is exposed to a second recombinase (Cre), resulting in the deletion of the NSI and the placement of the COIN in the direction felt for the transcription.

FIG. 8 illustrates an embodiment where, after treatment with recombinase (Flp exposure), the allele is exposed to a second recombinase, resulting in the inversion of COIN and NSI due to (alternative) placement of a recognition site of recombinase for the second recombinase in a 5 'position of NSI.

FIG. 9 illustrates the exon-intron structure of the mouse Hprtl gene in the region of exons 2 to 4, adapted from the mouse genome server Ensembl (top panel -
www.ensembl.org) and the region from exon 2 to exon 4 expands in the ECR browser (
http://ecrbrowser.dcode.org) to highlight conservation regions. Exon 3 is highlighted by an oval of points. The black vertical arrow indicates the insertion point of the drive and DSC sequence, while the gray arrow indicates the insertion point of the COIN element, all used to design the Hprt1MAF allele. Note that none of the evolutionarily conserved intronic sequences flanking exon 3 are interrupted in the resulting allele. The parallelogram of points indicates the region that will become the NSI in the Hprt1MAF allele.

FIG. 10 illustrates an example of an MFA, specifically the MFA for the Hprtl gene. Exon 3 plus evolutionarily conserved intronic sequences flanking exon 3 (as illustrated in FIG. 9) of Hprtl become the NSI. After targeting, the NSI is placed in the antisense chain with respect to the direction of the transcription of the Hprtl gene. A drive sequence - SA-lacZ-polyA - and a DSC are arranged upstream of the NSI. The drive sequence is arranged in the sense direction with respect to the direction of the transcription of the Hprtl gene, effectively acting as a gene trap element, and the repeal of the transcription downstream of the drive sequence. A COIN element is disposed downstream of the NSI in the antisense orientation with respect to the direction of the Hprtl gene transcription. Neither the COIN element nor the NSI can be incorporated into a productive Hprtl mRNA and, therefore, the resulting allele, Hprt1MFA, is a null allele with an informant (LacZ). The elements comprising the Hprt1MFA allele are flanked by site specific recombinase recognition sites arranged as follows: FRT- drive sequence-Rox-DSC-FRT3- (LoxP) - (NSI) - (Lox2372) - (FRT) - (FRT3) - (COIN) - (Lox2372) - (LoxP) -Rox, in which the parenthesis indicates the placement in the antisense orientation with respect to the direction of the transcription of the Hprt1 gene, or in the case of specific recombinase sites of the site with opposite orientation with respect to the mutually recognized pairs.

FIG. 11 illustrates an embodiment of an MFA, showing certain overlapping recombinant units (A) and resulting alleles that are generated by the action of a first recombinase (B) or a second (C) and third (D) recombinases.

FIG. 12 illustrates an embodiment of an MFA, which shows certain overlapping recombinant units (A) and resulting alleles that are generated by the action of a first recombinase (B) or a second (C) and third (D) recombinases.

FIG. 13 illustrates an embodiment of an MFA (A), which shows resulting alleles resulting from the action of a first recombinase that places the NSI in a sense orientation (B) and a second recombinase that places the NSI in an antisense orientation while placing the COIN in direction orientation (C).

FIG. 14 illustrates an embodiment of an MFA (A), which shows resulting alleles resulting from the action of a first recombinase that places the NSI in a sense orientation (B) and a second recombinase that places the NSI in an antisense orientation while placing the COIN in direction orientation (C).

FIG. 15 illustrates another embodiment of an MFA (A), which shows resulting alleles resulting from the action of a first recombinase that places the NSI in a sense orientation (B) and a second recombinase that places the NSI in an antisense orientation while placing the COIN in direction orientation (C).

FIG. 16 illustrates an example of an embodiment of MFA, where the informant is an SA (adml) -gtx-lacZ-pA, the DSC is Neo, the NSI is a critical exon (ec), and the COIN is Gtx-SA- HA-myc3-TM-T2A-GFP-pA (A), the placement of NSI in a direction oriented by the action of a recombinase while maintaining the COIN in antisense orientation (B) and, in addition, the cleavage of the NSI with the concomitant placement of the COIN in direction orientation (C); the arrows indicate primers used to confirm identities and orientations of recombinase sites in the MFA (A), and after treatment with recombinase (B and C).

FIG. 17 shows the results of cell viability and proliferation assays for Hprt1 + / Y, Hprt1MFA / Y, Hprt1C0IN / Y and Hprt1COIN'INV / Y ES cells, respectively, all grown in standard culture medium of

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ES cells either without 6-TG (without 6-TG; upper panels) or supplemented with a 6-TG 10 pM (6-TG, lower panels).

FIG. 18 shows Western blots of total protein preparations derived from Hprt1 + / Y (WT), Hprt1MFA / Y (MFA) cells - that is, the cells set target with the MFA of FIG. 16A, Hprt1C0IN / Y cells (MFA + FLPo) - that is, cells set as targets with the MFA of FIG. 16A and then treated with FLPo, and Hprt1C ° IN-NV / Y cells (MfA + FLPo + Cre) - that is, Hprt1COIN-INV / Y cells treated with Cre; the upper panel shows the detection of the Hprt1 protection, the central panel shows the detection of the LacZ (informant) protection and the lower panel shows the detection of the GAPDH protein as a load control.

SUMMARY

Methods and compositions are provided for making null and conditional alleles, and alleles that combine null and COIN features. In various embodiments, methods and compositions are provided for genetically modifying multifunctional alleles in a genome in a single target setting stage. Methods and compositions are provided for the complementary analysis of inactivation in genetically modified non-human animals, including methods comprising a single target fixation step.

Therefore, the present invention provides a nucleic acid construct, comprising:

(a) target fixing arms to direct the construction of nucleic acid to a target gene of a nucleic acid of a cell;

(b) a sense-oriented drive sequence with respect to transcription of the target gene, and a drug selection cassette (DSC) in sense or antisense orientation;

(c) in antisense orientation, a nucleotide sequence of interest (NSI) and a COIN (conditional element by inversion); Y

(d) recombinant units,

wherein the recombinant units comprise two first pairs of cogninate recombinase recognition sites recognized by a first recombinase, a first recombinant unit comprising the driving sequence, the DSC and the NSI, wherein the first recombinant unit is flanked by a pair of the first two pairs of recombinant kinase recognition sites recognized by the first recombinase, a second recombinant unit comprising the NSI, wherein the second recombinant unit is flanked by a pair of the first two pairs of recombinant kinase recognition sites recognized by the first recombinase, two second pairs of recombinant kinase recognition sites recognized by a second recombinase, and a third pair of recombinase recognition sites cognatos recognized by a third recombinase; wherein the first pairs, the second pairs and the third pairs of recombinase recognition sites are recognized by different recombinases;

wherein the recombinant units recombine after exposure to a first recombinase to form a conditional allele that lacks the drive sequence and the DSC and contains the NSI in a sense orientation and the COIN in antisense orientation; Y

wherein the conditional allele, when additionally exposed to the second recombinase, recombines to form an allele that lacks NSI and that has the COIN in a sense orientation.

The present invention also provides a method for modifying a non-human cell, comprising:

target a nucleotide sequence of interest in a cell with a nucleic acid construct of the invention to form a target cell, expose the target cell to the first recombinase to form the conditional allele and expose the conditional allele to a second recombinase that cleaves the NSI and places the COIN in a sense orientation.

The present invention also provides a nucleic acid construct, comprising:

a drug selection cassette (DSC), a reporter, a COIN, a nucleotide sequence of interest (NSI) and five pairs of recombinase sites arranged between the reporter, the DSC, the COIN and the NSI;

wherein no pair of recombinase sites is identical to any other pair, and where a first two pairs of recombinase sites are recognized by the same first recombinase, a second two pairs of recombinase sites are recognized by the same second recombinase and a fifth pair of recombinase sites is recognized by a third recombinase;

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where the first, second and third recombinases are not equal, and where with respect to the direction of transcription, the informant is in a sense orientation, the NSI is in antisense orientation, the COIN is in antisense orientation and the DSC is in sense or antisense orientation;

wherein the recombinase sites are arranged in recombinant units comprising (i) a first recombinant unit comprising the drive sequence, the DSC and the NSI, wherein the first recombinant unit is flanked by a pair of the first two pairs of recombinase recognition sites cognatos recognized by the first recombinase; and (ii) a second recombinant unit comprising the NSI, wherein the second recombinant unit is flanked by a pair of the first two pairs of cogninase recombinase recognition sites recognized by the first recombinase; Y

wherein the first two pairs and the second two pairs of recombinase sites are arranged so that upon exposure to the first recombinase, a modified allele is formed, wherein the first two pairs of recombinase sites direct the cleavage of the reporter, the cleavage of the DSC, the inversion of the NSI to sense orientation and the COIN is maintained in antisense orientation, and (a) the second pairs of recombinase sites are arranged so that, after exposure to the second recombinase, the NSI it is split and the COIN is placed in a direction oriented; or (b) the second two pairs of recombinase sites are arranged such that after exposure to the second recombinase, the NSI is placed in antisense orientation and the COIN is placed in a directional direction.

A modified allele is described, comprising a 3 'region of splicing and a splicing acceptor, a 3' driving sequence with respect to the splicing acceptor and a nucleotide sequence of interest (NSI) 3 ' with respect to the drive sequence, in which the NSI is in antisense orientation with respect to the target gene (or the locus being modified, or with respect to the drive sequence).

In one case, the drive sequence is selected from a microRNA, a transcription stop signal (such as a polyadenylation region), a nucleotide sequence encoding a cDNA, or any combination thereof, and may include regulatory elements. such as operators, enhancers and insulators. In a specific case, the cDNA encodes an informant (eg, encodes LacZ). In one case, the drive sequence comprises an exon. In a specific case, the exon is the 5 'most extreme exon of a locus.

In one case, the NSI comprises an exon. In one case, the NSI comprises an exon and a neighboring intronic sequence. In a specific case, the flanking exon is flanked at positions 5 'and 3' with an intronic sequence. In one case, the nucleotide sequence comprises two or more exons, and in a specific case, it comprises the intronic sequence (s). In another case, the NSI lacks an exon, or lacks a fragment of an exon.

In one case, the modified allele comprises a COIN. In one case, the COIN is in a 3 'position with respect to the NSI; otherwise, the COIN is in position 5 'with respect to the NSI.

In one case, the COIN is selected from an informant, a gene trap type element (GT type element) and a gene trap type informant (GT type informant). In a specific case, the GT type element is selected from cDNA-polyA of drug resistance SA. In a specific case, the GT-type reporter is selected from SA-reporter-polyA.

In one case, the COIN comprises a 3 'splice region. In a specific case, the 3 'splice region is followed by a sequence selected from a cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a small RNA, a codon jump element , an IRES, a polyadenylation sequence or any combination thereof. In a specific case, small RNA is a mirtron. In a specific case, the codon jump element is T2A, E2A or F2A.

In one case, the modified allele comprises a drug selection cassette (DSC).

In one case, the modified allele is in a target fixation construction comprising an upstream homology arm and a downstream homology arm. In one case, at least one homology arm is a mouse homology arm. In a specific case, the two homology arms are mouse homology arms.

In one case, the modified allele comprises, from 5 'to 3', a splice acceptor, a drive sequence, a DSC, an NSI and a COIN where the nucleotide sequence of interest and the COIN are both in antisense orientation with respect to the drive sequence, and five pairs of site specific recombinase recognition sites. In one case, the modified allele, upon exposure to a first site-specific recombinase that recognizes independently and reverses the sequence between a first pair of site-specific recombinase recognition sites and deletes a sequence between a second pair of site-specific recombinase recognition sites, results in an allele that comprises the NSI in a sense orientation for transcription, which lacks the DSC, and that comprises the COIN in antisense orientation. In one case, the modified allele comprises a third and fourth site specific recombinase recognition site arranged so that the additional exposure of the allele to a second recombinase that

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Independently recognizes the third and fourth site for specific site recombinase recognition resulting in the suppression of the NSI and the placement of the COIN in direction oriented for transcription.

In one aspect, the nucleic acid construct provided comprises (a) a reporter in a sense orientation and a DSC in a suitable orientation of choice, and in an antisense orientation a NSI and a COIN; (b) five pairs of site specific recombinase recognition sites, wherein the five pairs of recombinase recognition sites are recognized by no more than three recombinases; where after the treatment of the nucleic acid construction with a first recombinase, a modified allele is formed where (i) the NSI is arranged in a sense orientation, (ii) the COIN remains in antisense orientation, (iii) the reporter and the DSC and, (iv) the modified allele after treatment with a second recombinase suppresses the NSI and provides the COIN in a meaningful orientation.

In one embodiment, the five pairs of site specific recombinase recognition sites are the FRT3, Rox, FRT, loxP and lox2372 pairs.

In one embodiment, the first recombinase is a Flp recombinase, and the second recombinase is a Cre recombinase.

In one embodiment, the modified allele after treatment with the second recombinase results in an allele that cannot be deleted or reversed by the first or second recombinase.

In one embodiment, the nucleotide sequence of interest is a wild-type exon of a gene. In another embodiment, the NSI is an exon of a gene that has one or more nucleic acid substitutions, deletions or additions.

In one embodiment, the NSI is a wild type exon plus the intronic flanking of a gene. In another embodiment, the NSI is an exon plus the neighboring intronic sequence of a gene that has one or more nucleic acid substitutions, deletions or additions.

In one embodiment, the NSI is a wild type intron of a gene. In another embodiment, the NSI is an intron of a gene that has one or more nucleic acid substitutions, deletions or additions.

In one embodiment, the COIN comprises an exon or exons of a gene comprising one or more substitutions, deletions or additions of nucleic acids. In a specific embodiment, the COIN comprises an exon of a mammal. In a specific embodiment, the mammal is a human being, mouse, monkey or rat.

In one embodiment, the COIN comprises a 3 'region of splicing. In a specific embodiment, the 3 'region of splicing is followed by a selected sequence from a cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a small RNA, a codon jump element, a IRES, a polyadenylation sequence and a combination thereof. In a specific embodiment, the small RNA is a mirtron. In a specific embodiment, the codon jump element is a T2A.

In one embodiment, the COIN is selected from an informant, a gene trap type element (GT type element) and a gene trap type informant (GT type informant). In a specific embodiment, the GT type element is selected from cDNA-polyA of drug resistance SA. In a specific case, the GT-type reporter is selected from SA-reporter-polyA.

In one embodiment, the construction further comprises an upstream homology arm and a downstream homology arm. In one embodiment, the upstream homology arm and the downstream homology arm are mouse or rat homology arms. In a specific embodiment, the homology arms are mouse homology arms and the NSI comprises a human sequence. In a specific embodiment, the human sequence comprises a human exon that is a human homologue of a mouse exon.

In one embodiment, the reporter is selected from: a fluorescent protein, a luminescent protein or an enzyme. In a specific embodiment, the reporter is selected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed, MmGFP, luciferase, LacZ and alkaline phosphatase.

In one embodiment, the DSC comprises a sequence encoding a selected activity of neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror), blasticidine S deaminase (bsrr), xanthine / guanine phosphoribosyl transferase ( gpt), nourseotricin acetyltransferase (natl) and Herpes simplex thymidine kinase virus (HSV-tk).

In one aspect, the nucleic acid construct provided comprises an actuation sequence comprising a 3 'splice acceptor, followed by a sense oriented informant, DSC in a suitable choice orientation, an NSI in antisense orientation and a COIN in antisense orientation, where the drive sequence and the reporter are flanked upstream by a recombinase recognition site R1, a recombinase recognition site R2 is disposed between the informant and the DSC, a recombinase site R3 is disposed between the DSC and the nucleotide sequence of interest, an R4 recombinase site is disposed between the R3 and the NSI site, an R5 recombinase site is disposed between the NSI and the

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COIN, an R1 'recombinase site is disposed between the R5 site and the COIN, an R3' recombinase site is disposed between R1 'and the COIN, an R5' recombination site is disposed downstream of the COIN, a recombination site R4 'is disposed downstream of site R5' and a recombination site R2 'is disposed downstream of site R4'; where R1 and R1 'are in opposite orientation, R2 and R2' are in the same orientation, R3 and R3 'are in opposite orientation, r4 and R4' are in the same orientation and R5 and R5 'are in the same orientation.

In one embodiment, the informant is followed by a polyadenylation region.

In one embodiment, R1 and R1 'are recognized by a recombinase that recognizes R3 and R3'. In one embodiment, R4 and R4 'are recognized by a recombinase that recognizes R5 and R5'. In one embodiment, R2 and R2 'are not recognized by any recombinase that recognizes R1 / R1', R3 / R3 ', R4 / R4' or R5 / R5 '. In one embodiment, R1 and R1 ', R3 and R3' and R2 and R2 'are not recognized by any recombinase that recognizes R4 and R4' and R5 and R5 '. In one embodiment, R4 and R4 ', R5 and R5' and R2 and R2 'are not recognized by any recombinase that recognizes R1 and R1' and R3 and R3 '.

In one embodiment, treatment with a single recombinase results in a nucleic acid construct that lacks DSC, NSI and COIN. In a specific embodiment, the resulting nucleic acid construct consists essentially of the drive sequence, R1, and R2 or R2 '. In a specific embodiment, R1 is a FRT3 site and R2 (or R2 ') is a Rox site.

In one embodiment, treatment with a single recombinase results in a nucleic acid construct that comprises the sense-oriented drive sequence, but lacks DSC, lacks NSI and lacks COIN. In a specific embodiment, R2 and R2 'are Rox sites, and the single recombinase is Dre recombinase.

In one embodiment, treatment with a single recombinase results in a nucleic acid construct comprising the NSI in the antisense orientation and the COIN in antisense orientation. In one embodiment, the recombinase alone is a Flp recombinase, R1 and R1 'are a FRT variant sequence that does not cross-react with R3 and R3' (which are also FRT or FRT variants), R2 and R2 'are sequences of Rox, and R4 and R4 'are sequences of loxP or variant sequences of lox that do not cross-react with R5 and R5', where R5 and R5 'are variant sequences of lox.

In one embodiment, treatment with a single recombinase results in a nucleic acid construct comprising the NSI in a sense orientation and the COIN in antisense orientation. In one embodiment, the recombinase alone is a Flp, R1 and R1 'recombinase are sequences of FRT3, R2 and R2' are sequences of Rox, R3 and R3 'are sequences of FRT, R4 and R4' are sequences of loxP, R5 and R5 'are sequences of lox2372.

In one embodiment, the NSI is a wild type exon of a gene. In another embodiment, the NSI is an exon of a gene that has one or more nucleic acid substitutions, deletions or additions.

In one embodiment, the COIN comprises an exon or exons of a gene comprising one or more substitutions, deletions or additions of nucleic acids. In a specific embodiment, the COIN comprises an exon of a mammal. In one embodiment, the mammal is a human being, mouse, monkey or rat.

In one embodiment, the construction further comprises a homology arm upstream of the construction (an homology arm upstream) and a homology arm downstream of the construction (a homology arm downstream). In one embodiment, the upstream homology arm and the downstream homology arm are mouse or rat homology arms. In a specific embodiment, the homology arms are mouse homology arms and the NSI comprises a human sequence. In a specific embodiment, the human sequence comprises a human exon homologous to a mouse exon.

In one embodiment, the reporter is selected from: a fluorescent protein, a luminescent protein or an enzyme. In a specific embodiment, the reporter is selected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed, MmGFP, luciferase, LacZ and alkaline phosphatase. In one embodiment, the DSC comprises a sequence encoding a selected activity of neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror), blasticidine S deaminase (bsrr), xanthine / guanine phosphoribosyl transferase ( gpt), nourseotricin acetyltransferase (natl) and Herpes simplex thymidine kinase virus (HSV-tk).

In one aspect, the nucleic acid construct provided is a nucleic acid construct for the modification of a locus, comprising a first, second, third, fourth and fifth overlapping recombinant unit, wherein a recombinant unit includes a pair of sites of recognition of site specific recombinases cognatos, and where (a) the first recombinant unit is framed by recombinant sites R1 and R1 'in oppositional orientation (which allows inversion through R1 / R1'), where between R1 and R1 'are arranged in a directional direction sequence with respect to the direction of transcription of the target gene, followed by a recombinase site R2, followed by a DSC in a suitable orientation of choice, followed by a recombinase site R3 followed by an R4 recombinase site, followed by an NSI in antisense orientation, followed by an R5 recombinase site; (b) the second recombinant unit is framed by recombinase sites R2 and R2 'in the same orientation (allowing deletion through R2 / R2'), wherein between R2 and R2 'a DSC is arranged in a suitable orientation of choice, followed by R3, followed by R4, followed by NSI in antisense orientation, followed by R5 followed by R1 ', followed by the recombinase site R3' where R3 '

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it is in the opposite orientation with respect to R3 (allowing the inversion through R3 / R3 '), followed by a COIN in antisense orientation, followed by R5', where R5 'is in the same orientation with respect to R5, followed by R4 ', where R4' is in the same orientation with respect to R4, followed by R2 ', where R2' is in the same orientation with respect to R2 (allowing its deletion through R2 / R2 '); (c) the third recombinant unit is framed by the R3 and R3 'recombinase sites in opposite orientation (allowing the inversion through R3 / R3'), where between R3 and R3 'R4 are arranged, the NSI in antisense orientation , followed by R5, followed by R1 '; (d) the fourth recombinant unit framed by the recombinase sites R4 and R4 'in the same orientation, where between R4 and R4' the NSI is arranged in antisense orientation, followed by R5, followed by R1 ', followed by R3' , followed by COIN in antisense orientation, followed by R5 ', followed by R4'; and, (e) the fifth recombinant unit is framed by R5 and R5 'in the same orientation, where between R5 and R5' are arranged R1 'followed by R3' followed by the COIN in antisense orientation.

In one embodiment, R1 / R1 'and R3 / R3' are functional with respect to the same site specific recombinase, and said site specific recombinase is not functional with respect to R4 / R4 'and R5 / R5' and R2 / R2 '.

In one embodiment, R4 / R4 'and R5 / R5' are functional with respect to the same site specific recombinase, and said site specific recombinase is not functional with respect to R1 / R1 'and R3 / R3' and R2 / R2 '.

In one embodiment, R2 / R2 'are functional with a recombinase, wherein said recombinase is not functional with respect to any of R1 / R1', R3 / R3 ', R4 / R4' and R5 / R5 '.

In one embodiment, R1 / R1 'are FRT, FRT3, loxP or lox2372 sites. In one embodiment R3 / R3 'are FRT, FRT3, loxP or lox2372 sites. In one embodiment R4 / R4 'are FRT, FRT3, loxP or lox2372 sites. In one embodiment, R5 / R5 'are FRT, FRT3, loxP or lox2372 sites. In one embodiment, R2 / R2 'are Rox sites. In one embodiment, R2 / R2 'are attP / attB sites.

In a specific embodiment, R1 / R1 'and R3 / R3' are functional with a Flp recombinase. In another specific embodiment, R1 / R1 'and R3 / R3' are functional with a Cre recombinase.

In a specific embodiment, R4 / R4 'and R5 / R5' are functional with a Cre recombinase. In another specific embodiment, R4 / R4 'and R5 / R5' are functional with a Flp recombinase.

In one embodiment, R2 / R2 'are Rox sites that are functional with a Dre recombinase. In another embodiment, R2 / R2 'are attP / attB sites that are functional with PhiC31 integrasa (phiC31 \ int).

In one embodiment, the reporter is selected from: a fluorescent protein, a luminescent protein or an enzyme. In a specific embodiment, the reporter is selected from GFP, eGFP, cFp, YFP, eYFP, BFP, eBFP, DsRed, MmGFP, luciferase, LacZ and alkaline phosphatase.

In one embodiment, the DSC comprises a sequence encoding a selected activity of neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror), blasticidine S deaminase (bsrr), xanthine / guanine phosphoribosyl transferase ( gpt), nourseotricin acetyltransferase (natl) and Herpes simplex thymidine kinase virus (HSV-tk).

In one embodiment, the NSI is a wild type exon of a gene. In another embodiment, the NSI is an exon of a gene that has one or more nucleic acid substitutions, deletions or additions.

In one embodiment, the COIN comprises an exon of a gene comprising one or more nucleic acid substitutions, deletions or additions. In a specific embodiment, the COIN comprises an exon of a human being, mouse, monkey or rat.

In one embodiment, the COIN comprises a 3 'region of splicing. In a specific embodiment, the 3 'region of splicing is followed by a selected sequence from a cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a small RNA, a codon jump element, a IRES, a polyadenylation sequence and a combination thereof. In a specific embodiment, the small RNA is a mirtron. In a specific embodiment, the codon jump element is T2A, E2A or F2A.

In one embodiment, the COIN is selected from an informant, a gene trap type element (GT type element) and a gene trap type informant (GT type informant). In a specific embodiment, the GT type element is selected from cDNA-polyA of drug resistance SA. In a specific case, the GT-type reporter is selected from SA-reporter-polyA.

In one embodiment, the construction further comprises a homology arm upstream of the construction (an homology arm upstream) and a homology arm downstream of the construction (a homology arm downstream). In one embodiment, the upstream homology arm and the downstream homology arm are mouse or rat homology arms. In a specific embodiment, the homology arms are mouse homology arms and the NSI comprises a human sequence. In a specific embodiment, the human sequence comprises a human exon that is a homologue of a mouse exon.

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A multifunctional allele is described which comprises two or more recombinant units that are recognized by two or more different recombinases, each of the recombinant units being defined by a pair of compatible recombinase recognition sites that define the limits of the recombinant unit. Each of the recombinant units comprises one or more internal recombinase recognition sites. The one or more internal recombinase recognition sites are selected such that, after recombination by a first recombinase of a recombinant unit of the multifunctional allele, the one or more internal recombinase recognition sites within a recombinant unit are then paired with one or more internal recombinase units within another recombinant unit to allow inversion and / or deletion by the first recombinase of a sequence that extends to two or more recombinant units of the multifunctional allele, where inversion and / or deletion is possible only after the inversion of the one or more internal recombinase recognition sites.

In one case, the inversion and / or deletion is accompanied by the inversion of an additional recombinase recognition site of the multifunctional allele, where the inversion of the additional recombinase recognition site allows the inversion or deletion of an element of the multifunctional allele by a second recombinase.

A multifunctional allele is described, comprising: (a) a first, a second, a third, a fourth and a recombinant fifth unit, wherein each of the recombinant units is limited by compatible recombinase recognition sites and wherein the first recombinant unit overlaps with the second recombinant unit, and wherein the third, fourth and fifth recombinant units are contained within the second recombinant unit; (b) a first recombinant unit comprising a 3 'splice acceptor and a splice region operatively linked to a drive sequence, a DSC, and an NSI; (c) a second recombinant unit comprising the DSC, the NSI and a COIN; (d) a third recombinant unit comprising the NSI; (e) a fourth recombinant unit comprising the NSI and the COIN; (f) a fifth recombinant unit comprising the COIN; wherein the multifunctional alleles comprise a first pair of recombinase recognition sites that flank the first recombinant unit upstream and downstream that allow a first inversion of the first recombinant unit, where the first investment results in a second inversion of a recombinase site within the second recombinant unit, wherein the second inversion directs the recombinase site within the second recombinant unit in order to suppress the drive sequence and suppress the DSC.

In one case, a single recombinase recognizes the first pair of recombinase recognition sites and also suppresses the drive sequence and drug selection cassette.

In one case, the second inversion guides a recombination site so that after the inversion a second set of recombinase recognition sites is formed that allows the deletion of the NSI and / or COIN inversion.

In one aspect, the nucleic acid construct provided comprises an MFA comprising, from 5 'to 3' with respect to the direction of transcription, a COIN in antisense orientation, an NSI in antisense orientation, a DSC and a reporter in orientation. sense, where after the treatment of the MFA with a selected recombinase, the COIN, the NSI and the DSC are cleaved and the informant remains in a sense orientation; and where after an alternative treatment with a different selected recombinase, the reporter and the DSC are cleaved, the COIN remains in antisense orientation and the NSI is arranged in a sense orientation, so that after further treatment with yet another different selected recombinase, the NSI is split and the COIN is arranged in a meaningful orientation.

In one embodiment, the MFA comprises a first recombinant unit, a second recombinant unit and a third recombinant unit, wherein the first recombinant unit overlaps the second and third recombinant unit, and wherein the second recombinant unit overlaps the first and third recombinable unit.

In one embodiment, the first recombinant unit comprises a COIN in reverse orientation (antisense) and an NSI in reverse orientation, wherein the recombinant unit is flanked upstream of the COIN and downstream of the NSI by the compatible recombinase sites R2 and R2 'oriented to direct a deletion; the second recombinant unit overlaps the first recombinable unit, and the second recombinable unit is recombinable by the action of a recombinase at a recombination site upstream of the DSC and a recombination site downstream of the reporter, where the recombination sites they are oriented to direct an inversion, and where the recombination site upstream of the DSC is followed by a sequence comprising the NSI. In a specific embodiment, the MFA comprises, from 5 'to 3' with respect to the orientation in a sense chain, a first site of recombinase R1, a second site of recombinase R2, a third site of recombinase R3, the COIN in orientation antisense, a fourth R4 recombinase site, a fifth R5 recombinase site, a sixth R3 'recombinase site that is compatible with R3 and is oriented to direct a deletion of the sequence between R3 and R3', the NSI in antisense orientation, a seventh R2 'recombinase site that is compatible with R2 and is oriented to direct a deletion of the sequence between R2 and R2', an eighth R4 'recombinase site, a DSC, a ninth R1' recombinase site that is compatible with R1 and is oriented to direct a deletion of the sequence between R1 and R1 ', a sense oriented informant and a tenth recombinase site R5' that is compatible with R5 and is oriented to direct a reversal of the sequence between R5 and R5 ' .

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In a specific embodiment, R1 / R1 'are Rox sites, R2 / R2' are loxP sites, R3 / R3 'are lox2372 sites, R4 / R4' are FRT sites and R5 / R5 'are FRT3 sites. In a specific embodiment, the MFA comprises an arrangement of recombinase sites and COIN, NSI, DSC and the reporter as shown in FIG. 11, Panel A. In a specific embodiment, after exposure to a single recombinase that recognizes R1 / R1 ', an allele is formed as shown in FIG. 11, Panel B. In a specific embodiment, after exposure to a single recombinase that recognizes R4 / R4 'and R5 / R5', an allele is formed as shown in FIG. 11, Panel C. In a specific embodiment, after the exposure of the allele of FIG. 11, Panel C to an additional recombinase that recognizes R2 / R2 'and R3 / R3', an allele is formed as shown in FIG. 11, Panel D.

In one aspect, the nucleic acid construct provided comprises an MFA comprising, from 5 'to 3' with respect to the direction of transcription, an NSI in antisense orientation, a DSC, a reporter in sense orientation and a COIN in orientation. antisense; wherein after the treatment of the MFA with a selected recombinase, the NSI and the DSC are cleaved, the informant remains in a sense orientation and the COIN is maintained in an antisense orientation; and where after an alternative treatment with a different selected recombinase, the DSC and the reporter are cleaved, and the NSI is disposed in a sense orientation and the COIN is in antisense orientation, and where after the alternative treatment with the different selected recombinase, the allele is treated with yet another different selected recombinase, resulting in the cleavage of NSI and the disposition of the COIN in a sense orientation.

In one embodiment, the MFA comprises a first recombinant unit, a second recombinant unit and a third recombinant unit, wherein the first recombinant unit overlaps the second and third recombinant unit, and wherein the second recombinant unit overlaps the first and third recombinable unit. In one embodiment, the first recombinant unit comprises a DSC and a sense-oriented reporter, wherein the recombinant unit is flanked upstream of the DSC by recombination sites R2 followed by R3, and is flanked downstream of the reporter by recombinant site R3 ', where R2 / R3' are oriented to direct an investment, and where the DSC is preceded by R2 'oriented with respect to R2 to direct an investment; the second recombinant unit is flanked, upstream of the antisense NSI, by R4 and is flanked, downstream of the antisense COIN, by R4 ', where R4 / R4' are oriented to direct a cleavage, and where the second recombinable unit includes the DSC and informant; and the third recombinable unit is flanked upstream by R1 and downstream by R1 ', where R1 / R1' are oriented to direct a split, where upstream and adjacent to R1 'is the DSC and where downstream and adjacent to R1 is R2. In a specific embodiment, the MFA comprises, from 5 'to 3' with respect to the direction of the transcription, R1, R2, R3, R4, the NSI in antisense orientation, R5, R2 ', where R2 / R2' are oriented to direct an investment, the DSC, R1 ', where R1 / R1' are oriented to direct an investment, the reporter gene, R3 ', where R3 / R3' are oriented to direct an investment, the COIN in antisense orientation , R5 ', where R5 / R5' are oriented to direct a split, and R4 ', where R4 / R4' are oriented to direct a split.

In a specific embodiment, R1 / R1 'are Rox sites, R2 / R2' are FRT or FRT3 sites, R3 / R3 'are FRT or FRT3 sites, which are not the same as R2 / R2', R4 / R4 'are sites lox2372 or loxP sites, and R5 / R5 'are lox2372 sites or loxP sites that are not the same as R4 / R4'.

In a specific embodiment, the MFA comprises an arrangement of recombinase sites and COIN, NSI, DSC and the reporter as shown in FIG. 12, Panel A. Treatment with a selected recombinase results in the allele shown in FIG. 12, Panel B. Alternative treatment with a different selected recombinase results in the allele shown in FIG. 12, Panel C. The treatment of the allele of FIG. 12, Panel C with yet another different recombinase results in the allele shown in FIG. 12, Panel D.

In one aspect, the nucleic acid construct provided comprises an MFA comprising, from 5 'to 3' with respect to the direction of transcription, a reporter in a sense orientation, a DSC, an NSI in antisense orientation and a COIN in orientation. antisense; in that after the treatment of the MFA with a first recombinase selected, the informant is cleaved, the NSI is arranged in a sense orientation and the COIN remains in antisense orientation, and where the allele comprises recombinase sites that allow a sequence inversion, that after the treatment with a second recombinase selected, it would place the COIN in a sense orientation and the NSI in an antisense orientation. In one embodiment, following the first recombinase selected, the allele is treated with the second recombinase selected. In one embodiment, the COIN notes that the NSI has been arranged in antisense orientation after treatment with the second recombinase.

In one embodiment, the MFA comprises, from 5 'to 3' with respect to the direction of transcription, an R1 recombinase site, a reporter, a second R2 recombinase site, a DSC, a third R3 recombinase site, a NSI in antisense orientation, a fourth R4 recombinase site, a fifth R5 recombinase site, a sixth R1 'recombinase site that is compatible with R1 and that is oriented with respect to R1 to direct an inversion, a seventh R3 recombinase site 'that it is compatible with R3 and that it is oriented with respect to R3 to direct an investment, a COIN in antisense orientation, an eighth site of recombinase R5' that is compatible with R5 and that is oriented with respect to R5 to direct a cleavage, a ninth R4 'recombinase site that is compatible with R4 and that is oriented with respect to R4 to direct a cleavage and a tenth recombinase site R2' that is compatible with R2 and that is oriented with respect to R2 to direct a cleavage. In a specific embodiment, R1 / R1 'are FRT3 or FRT sites, R2 / R2' are Rox sites, R3 / R3 'are FRT3 or FRT sites, which are

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different from R1 / R1 ', R4 / R4' are loxP or lox2372 sites and R5 / R5 'are loxP or lox2372 sites that are different from R4 / R4' sites.

In a specific embodiment, the MFA comprises an arrangement of recombinase and COIN, NSI, DSC and reporter sites as shown in FIG. 13, Panel A. Treatment with a selected recombinase results in the allele shown in FIG. 13, Panel B. Treatment of the allele of FIG. 13, Panel B with a different recombinase results in the allele shown in FIG. 13, Panel C.

In one aspect, the nucleic acid construct provided comprises an MFA comprising, from 5 'to 3' with respect to the direction of transcription, a COIN in antisense orientation, an NSI in antisense orientation, a DSC and a reporter in orientation. sense; in that after the treatment of the MFA with a first recombinase selected, the reporter is cleaved, the NSI is arranged in a sense orientation, and the COIN remains in antisense orientation, and where the allele comprises recombinase sites that allow a sequence inversion that, after treatment with a second recombinase selected, the COIN would be available in a sense orientation and the NSI in antisense orientation. In one embodiment, following the first recombinase selected, the allele is treated with the second recombinase selected. In one embodiment, the COIN notes that the NSI has been arranged in antisense orientation after treatment with the second recombinase.

In one embodiment, the MFA comprises, from 5 'to 3' with respect to the direction of transcription, a recombinase site R1, a second recombinase site R2, a third recombinase site R3, a COIN in antisense orientation, a fourth R4 recombinase site, a fifth R5 recombinase site, a sixth R3 'recombinase site that is compatible with R3 and that is oriented with respect to R3 to direct a cleavage, a seventh recombinase site R2' that is compatible with R2 and that it is oriented with respect to R2 to direct a cleavage, an NSI in antisense orientation, an eighth R4 'recombinase site that is compatible with R4 and that is oriented with respect to R4 to direct an inversion, a DSC, a ninth site of R1 'recombinase that is compatible with R1 and that is oriented with respect to R1 to direct a cleavage, a sense-oriented reporter and a tenth recombinase site R5' that is compatible with R5 and that is oriented with respect to R5 to direct Make an investment. In a specific embodiment, R1 / R1 'are Rox sites, R2 / R2' are loxP or lox2372 sites, R3 / R3 'are loxP or lox2372 sites that are different from R2 / R2', R4 / R4 'are FRT or FRT3 sites and R5 / R5 'are FRT or FRT3 sites that are different from R4 / R4'.

In a specific embodiment, the MFA comprises an arrangement of recombinase sites and COIN, NSI, DSC and the reporter as shown in FIG. 14, Panel A. Treatment with a selected recombinase results in the allele shown in FIG. 14, Panel B. Treatment of the allele of FIG. 14, Panel B with a different recombinase results in the allele shown in FIG. 14, Panel C.

In one aspect, the nucleic acid construct provided comprises an MFA comprising, from 5 'to 3' with respect to the direction of transcription, an NSI in antisense orientation, a DSC, a reporter in sense orientation and a COIN in orientation. antisense; in which after the treatment of the MFA with a first recombinase selected, the informant is cleaved, the DSC is cleaved, the NSI is arranged in a sense orientation and the COIN is maintained in an antisense orientation, and where after the treatment with the first selected recombinase The allele comprises recombinase sites that allow an inversion of the sequence that after treatment with a second selected recombinase would have the COIN in a sense orientation and the NSI in an antisense orientation. In one embodiment, following the first recombinase selected, the allele is treated with the second recombinase selected. In one embodiment, the COIN notes that the NSI has been arranged in antisense orientation after treatment with the second recombinase.

In one embodiment, the MFA comprises, from 5 'to 3' with respect to the direction of transcription, a recombinase site R1, a second recombinase site R2, a third recombinase site R3, an NSI in antisense orientation, a fourth R4 recombinase site, a fifth R5 recombinase site, a sixth R2 'recombinase site that is compatible with R2 and that is oriented with respect to R2 to direct an inversion, a DSC, a seventh R1' recombinase site that is compatible with R1 and that is oriented with respect to R1 to direct a split, a reporter in a sense orientation, an eighth site of recombinase R3 'that is compatible with R3 and that is oriented with respect to R3 to direct an investment, a COIN in reverse orientation, a ninth R5 'recombinase site that is compatible with R5 and that is oriented with respect to R5 to direct a cleavage and a tenth recombinase site R4' that is compatible with R4 and that is oriented with respect to R4 to directa split In a specific embodiment, R1 / R1 'are Rox sites, R2 / R2' are FRT or fRT3 sites, R3 / R3 'are FRT or FRT3 sites that are different from R2 / R2', R4 / R4 'are loxP or lox2372 sites and R5 / R5 'are loxP or lox2372 sites that are different from R4 / R4'.

In a specific embodiment, the MFA comprises an arrangement of recombinase sites and COIN, NSI, DSC and the reporter as shown in FIG. 15, Panel A. Treatment with a selected recombinase results in the allele shown in FIG. 15, Panel B. Treatment of the allele of FIG. 15, Panel B with a different recombinase results in the allele shown in FIG. 15, Panel C.

A multifunctional allele is described, comprising a DSC, a reporter, a COIN, an NSI and five pairs of recombinase sites arranged between the reporter, the DSC, the COIN and the NSI, where no pair of sites of

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recombinase is identical to any other pair, and where a first two pairs of recombinase sites are recognized by the same first recombinase, a second two pairs of recombinase sites are recognized by the same second recombinase and the fifth pair of recombinase sites are recognized by a third recombinase, where the first, second and third recombinase are not identical, and where, with respect to the direction of transcription, the MFA comprises (from 5 'to 3'): (a) a sequence of actuation (eg, with informer) in direction direction, DSC in direction sense or antisense, NSI in orientation antisense, COIN in orientation antisense; (b) the COIN in antisense orientation, the NSI in antisense orientation, the DSC in sense or antisense orientation, the informer in sense orientation; (c) the NSI in antisense orientation, the DSC in sense or antisense orientation, the informer in sense orientation, the COIN in antisense orientation; (d) the informant in a meaningful orientation, the DSC in a sense or antisense orientation, the NSI in antisense orientation, the COIN in antisense orientation; (e) the COIN is in antisense orientation, the NSI is in antisense orientation, the DSC is in a sense or antisense orientation, the informant is in a meaningful orientation; or (f) the NSI in antisense orientation, the DSC in sense or antisense orientation, the informer in sense orientation, the COIN in antisense orientation.

In one case, the arrangement is as in (a), and the pairs of recombinase sites are arranged such that upon exposure to the third recombinase, the fifth pair of recombinase sites directs a cleavage of the DSC, the NSI and the COIN, where the informant remains in a meaningful orientation.

In one case, the arrangement is as in (a), and the pairs of recombinase sites are arranged such that upon exposure to the first recombinase, a modified MFA is formed, where the first two pairs of recombinase sites direct the excision of the informant and the excision of the DSC and the investment of the NSI to a meaningful orientation, where the COIN remains in antisense orientation. In a further case, the modified MFA comprises the second two pairs of recombinase sites that, upon exposure to the second recombinase, result in an allele where the NSI is cleaved and the COIN is disposed in the sense orientation.

In one case, the arrangement is as in (b), and the pairs of recombinase sites are arranged such that after exposure to the third recombinase, the fifth pair of recombinase sites directs a cleavage of the COIN, the NSI and the DSC, where the informant remains in a meaningful direction.

In one case, the arrangement is as in (b), and the pairs of recombinase sites are arranged such that after exposure to the first recombinase, a modified MFA is formed where the first two pairs of recombinase sites direct the splitting of the DSC and the informer and the direct investment of the NSI to the meaningful orientation, where the COIN remains in antisense orientation. In a further case, the modified MFA comprises the second two pairs of recombinase sites that, after exposure to the second recombinase, give rise to an allele where the NSI is cleaved and the COIN is placed in the direction of orientation.

In one case, the arrangement is as in (c), and the pairs of recombinase sites are arranged such that after exposure to the fifth recombinase, the NSI and the DSC are cleaved and the reporter and the COIN are kept in antisense orientation.

In one case, the arrangement is as in (c), and the recombinase pairs or sites are arranged such that upon exposure to the first recombinase, a modified MFA is formed where the DSC and the reporter are cleaved, and the NSI is arranged in a meaningful orientation, where the COIN is maintained in an antisense orientation. In a further case, the modified MFA comprises the second two pairs of recombinase sites that, upon exposure to the second recombinase, result in an allele where the NSI is cleaved and the COIN is disposed in a sense orientation.

In one case, the arrangement is as in (d), and the pairs of recombinase sites are arranged such that after exposure to the fifth recombinase, the DSC, the NSI and the COIN are cleaved and the reporter is kept in direction orientation.

In one case, the arrangement is as in (d), and the pairs of recombinase sites are arranged such that upon exposure to the first recombinase, a modified MFA is formed where the reporter and the DSC are cleaved, and The NSI is arranged in a meaningful orientation, where the COIN is maintained in an antisense orientation. In a further case, the modified MFA comprises the second two pairs of recombinase sites that, upon exposure to the second recombinase, result in an allele where the COIN is disposed in a sense orientation and the NSI is disposed in an antisense orientation.

In one case, the arrangement is as in (e), and the pairs of recombinase sites are arranged such that after exposure to the fifth recombinase, the COIN, the NSI and the DSC are cleaved and the reporter is kept in direction orientation.

In one case, the arrangement is as in (e), and the pairs of recombinase sites are arranged such that after exposure to the first recombinase, a modified MFA is formed where the DSC and the reporter are cleaved, the NSI is arranged in a meaningful orientation and the COIN is maintained in an antisense orientation. In a further case, the modified MFA comprises the second two pairs of recombinase sites arranged in such a manner.

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that, after exposure to the second recombinase, the NSI is placed in antisense orientation and the COIN is placed in the sense orientation.

In one case, the arrangement is as in (f), and the pairs of recombinase sites are arranged in such a way that upon exposure to the fifth recombinase, the NSI and the DSC are cleaved, the reporter remains in a directional direction and The COIN remains in antisense orientation.

In one case, the arrangement is as in (f), and the pairs of recombinase sites are arranged such that upon exposure to the fifth recombinase, the NSI and the DSC are cleaved and the reporter is maintained in a directional direction and COIN remains in antisense orientation.

In one case, the arrangement is as in (f), and the pairs of recombinase sites are arranged such that upon exposure to the first recombinase, a modified MFA is formed where the DSC and the reporter are cleaved and the NSI is arranged in a meaningful orientation and the COIN is maintained in an antisense orientation. In a further case, the modified MFA comprises the second two pairs of recombinase sites arranged in such a way that, after exposure to the second recombinase, the NSI is arranged in antisense orientation and the COIN is arranged in a sense orientation.

A method for producing a cell comprising a construction having a nucleotide sequence of interest in antisense orientation and a COIN in antisense orientation is described, comprising the step of introducing an MFA into a genome of a cell as described herein. memory, identify the cell comprising the MFA, followed by a stage of exposing the genome to a first recombinase, in which the action of the first recombinase on the construction in the genome results in the nucleotide sequence of interest that is placed in the direction orientation.

In one case, the cell is a pluripotent cell, an induced pluripotent cell, a totipotent cell or an ES cell. In a specific case, the ES cell is a mouse or rat ES cell.

In one case, the construction is introduced into the cell by homologous recombination. In another case, the construct is randomly integrated into a nucleic acid of the cell. In one embodiment, the nucleic acid of the cell is the genome of the cell.

In one case, the NSI comprises an exon. In one case, the NSI comprises an exon and an intronic flanking sequence. In a specific case, the flanking exon is flanked at positions 5 'and 3' with the intronic sequence. In one case, the nucleotide sequence comprises two or more exons, and in a specific case it comprises the intronic sequence (s). In another case, the NSI lacks an exon, or lacks a fragment of an exon.

In one case, NSI is a wild-type exon or exons of a gene. In another case, NSI is an exon or exons of a gene that has one or more substitutions, deletions or additions of nucleic acids.

In one case, the COIN comprises an exon of a gene comprising one or more nucleic acid substitutions, deletions or additions. In a specific case, the COIN comprises an exon of a human, mouse, monkey or rat gene.

In one case, the COIN comprises a 3 'region of splicing. In a specific embodiment, the 3 'region of splicing is followed by a selected sequence from a cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a small RNA, a codon jump element, a IRES, a polyadenylation sequence and a combination thereof. In a specific case, small RNA is a mirtron. In a specific case, the codon jump element is T2A, E2A or F2A.

In one case, the COIN is selected from an informant, a gene trap type element (GT type element) and a gene trap type informant (GT type informant). In a specific embodiment, the GT type element is selected from cDNA-polyA of drug resistance SA. In a specific embodiment, the GT-type reporter is selected from SA-reporter-polyA.

A method for arranging a multifunctional allele in a mouse cell genome is described, comprising a step of introducing a target fixation construct into a locus into a mouse cell, comprising a first recombinant unit comprising (a) a drive sequence (e.g., a nucleotide sequence and / or an informer); (b) a DSC; (c) an NSI in antisense orientation with respect to the locus; (d) a COIN in antisense orientation with respect to the locus; and (e) site-specific recombinase recognition sites arranged in recombinant units to suppress the informant and the DSC, to reverse the NSI back to the sense orientation, and to reverse the COIN and suppress or reverse the NSI.

In one case, the site-specific recombinase recognition sites are arranged in recombinant units so that the NSI will be reverted to the antisense chain and the COIN will be reversed to the sense chain. In another case, the site-specific recombinase recognition sites are arranged in recombinant units so that the NSI will be suppressed and the COIN will be inverted to the sense chain.

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In one case, the recombinant units are arranged in such a way that upon exposure of the MFA-modified target locus to a first recombinase, a first recombinant unit comprising the reporter and DSC is suppressed and the NSI is arranged in the sense direction with respect to to the locus and the COIN remains in the antisense orientation, forming a second recombinant unit.

In one case, the nucleotide sequence of interest in the antisense orientation is an exon in the antisense orientation, or an exon flanked by the intronic sequence, where the exon and the intronic sequence are each in antisense orientation. In a specific case, the exon that is provided in the antisense orientation is identical to the exon that is being replaced by the target setting construction. In a specific case, the NSI in antisense orientation is an exon and the sequence surrounding the exon. In a specific case, the NSI is two or more exons. In a specific embodiment, the NSI is a non-exotic sequence.

In one case, the second recombinant unit generated by the action of the first recombinase is exposed to a second recombinase, where the second recombinase suppresses the NSI and arranges the COIN in the sense orientation.

In one case, the second recombinant unit generated by the action of the first recombinase is exposed to a second recombinase, where the second recombinase arranges the NSI in antisense orientation and arranges the COIN in a sense orientation.

A method for complementing an inactivation is also described, which comprises introducing an MFA into a non-human animal as described herein, wherein the nucleic acid construct comprises a wild-type nucleic acid sequence in antisense orientation and a COIN in the antisense orientation, in which after exposure of the nucleic acid construct to a first recombinase the wild-type nucleic acid sequence is inverted to detect the orientation and is transcribed, but the COIN remains in the antisense orientation; and where, after exposure to a second recombinase, the wild-type nucleic acid sequence is cleaved, or the antisense chain is inverted again, and the COIN is inverted to detect orientation.

In one case, the non-human animal is a mouse.

In one case, the COIN is an informative element. In one embodiment, the reporter element is selected from a fluorescent protein, a luminescent protein or an enzyme. In a specific case, the reporter is selected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed, MmGFP, luciferase, LacZ, and alkaline phosphatase.

A mammalian cell comprising a multifunctional allele according to the invention is also described.

In one case, the mammalian cell is selected from a mouse cell and a rat cell. In one case, the cell is selected from a stem cell, an embryonic stem cell (ES), an induced pluripotent cell, a pluripotent cell and a totipotent cell.

Also described is a non-human embryo or non-human animal comprising a multifunctional allele according to the invention.

In one case, the non-human or non-human animal embryo comprises a multifunctional allele that has been exposed to one or more site specific recombinases. In a specific case, the multifunctional allele has been exposed to one or more site specific recombinases as a result of a culture stage where a non-human animal comprising a multifunctional allele has been coupled to a non-human animal comprising one or more site specific recombinases, and the non-human embryo or the non-human animal is a progeny of the stage of crfa.

Also described is a cell comprising an MFA as described herein, wherein the cell is a mammalian cell, e.g. eg, an ES cell or an induced pluripotent or pluripotent cell. In a specific case, the cell is a mouse or rat cell.

A non-human animal is also described, which comprises an MFA as described herein, or an MFA that has been exposed to one or more recombinases as described herein.

A non-human embryo is also described, which comprises an MFA as described herein, or an MFA that has been exposed to one or more recombinases as described herein.

A cell, a non-human embryo or a non-human animal produced using an MFA as described herein is also described.

A cell, a non-human embryo or a non-human animal produced using an MFA as described herein is also described.

Any aspect or embodiment may be used in relation to any other aspect or embodiment, as appropriate, p. For example, any informant or DSC resigned in connection with any particular MFA performance will be

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it can be used with any embodiment of MFA described herein, and any particular recombinase or recombinase site mentioned in connection with any particular embodiment of MFA can be used with any embodiment of MFA described herein.

Other embodiments are described and will be apparent to those skilled in the art from a review of the following detailed description.

DETAILED DESCRIPTION

The invention is not limited to particular methods, and the experimental conditions described, since such methods and conditions may vary. The terminology used herein is for the purpose of describing only particular embodiments, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

Unless otherwise defined, all technical and scientific terms and expressions used herein have the same meaning as that commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or assay of the present invention, particular methods and materials are now described.

The phrase "sense orientation," or "sense" refers to the direction of coding or sense chain of a transcribable nucleic acid sequence in the local genome context, e.g. eg, when a sequence is arranged in "sense orientation" in or near a transcribable sequence in a genome, the sequence orientation is compatible with transcription and, for genes encoding proteins, also the translation of the sequence into the region or locus or gene in which the sequence is arranged. The phrase "antisense orientation" or "antisense" refers to the placement of a sequence in a region or locus or gene in which the sequence is in the opposite (or antisense) chain to which it is compatible with the transcription. Thus, in a specific example, if a sequence is arranged in "sense" orientation in a gene, it can generally be transcribed. If a sequence is arranged in "antisense" orientation, it will usually not be transcribed. For loci where the transfer between the sense and antisense chains could result in the transcription of any of the strands, the sequence can be selected or treated by genetic engineering such that the transfer of meaning to antisense or antisense to sense results in the transcription of a string, but not either.

The term "COIN" includes the reference to a conditional element. A conditional element comprises a nucleotide sequence, whose expression (or failure to express) is conditioned by the occurrence of an independent event. For example, a coding region that in a sense orientation would encode a protein or fragment thereof or a non-coding RNA (cRNA) is arranged in the antisense orientation, flanked on both sides by site specific recombination sites in the opposite orientation. In the absence of a site specific recombinase that recognizes flanking sites, the coding region is not transcribed, as it is disposed in the antisense chain with respect to the target gene. After treatment with the specific recombinase of the cognitive site, the COIN sequence is reversed and, as a result, it is incorporated into the transcribed message, resulting in the expression of the protein or fragment thereof or in that of the RNA.

The term "incompatible", when used to describe two or more recombinase recognition sites, refers to the quality that two or more recombinase recognition sites cannot recombine between each other (but the two or more recombinase recognition sites they can be recombined with other recombinase (eg, identical) cognate recognition sites.

Inactivations and Conditional Alleles

The study of gene function by genetic methods has been based on the discovery of naturally occurring variants or mutant alleles, or on the deliberate generation of such variants and mutant alleles. The latter has proceeded either by random mutagenesis followed by traces based on the phenotype and then elucidation of the causal mutation - a process referred to as "direct genetics" or through the methodology of genetic engineering by which mutations are perform on specific genes or "target" loci - an approach referred to as "reverse genetics".

In the mouse - the most used mammalian model organism - the ability to deal with genetically engineered specific mutations, molecularly extremely well defined through gene targeting, has dominated the field of inverse genetics. However, most of the variants made to date have been relatively simple null alleles, commonly known as "inactivated alleles" or simply "inactivations," and usually encompass a deletion of the exon-intron region of a gene or part thereof. , and in more recent years with the concomitant substitution of that region with an informative cDNA, such as LacZ. The adaptation of site-specific recombinase and its cognitive recognition sites (such as Cre / lox, Dre / Rox, PhiC31 \ int / attP-attB, Flp / FRT), bacteriophage or yeast derivatives and modified for use in mammals, is a more recent development that has not only made possible the post-fixation excision as a target of the DSC (insofar as it is flanked by site specific recombinase recognition sites), but has also allowed treatment by genetic engineering of conditional null alleles. They have

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developed conditional null alleles in which the exon-intron region of the target gene - or more frequently a part of it - is flanked by recombinase recognition sites, making the modified alleles susceptible to conversion to the null state by action of cognata recombinase. The advantage of this method over regular inactivations is that the conversion of the modified gene into an inactivation can be controlled space-temporarily by controlling the place (organ, tissue or cell type), time and, sometimes, also the duration that cognata recombinase will be active.

Traditionally, null conditional alleles have been treated by genetic engineering as a follow-up of the corresponding simple inactivated alleles, especially in cases where the latter is lethal embryonic and / or shows a plurality of phenotypes, therefore, making it impossible the study of the function of the target gene in an adult environment (in the case of embryonic lethality), or difficult to interpret in a specific cell type or biological process (in the case where the gene shows a plurality of phenotypes). Given the amount of effort, time and money needed to generate genetically modified mice through gene targeting, this step-by-step way of first generating an inactivation has been considered burdensome, then deciphering its phenotype and then treating by engineering genetics a conditional null by an increasing number of researchers. In addition, for a small number of genes, regular inactivated alleles cannot pass through the germ line, since they are the result of embryonic lethality even in the null heterozygous state. Therefore, the desire to be able to treat dual alleles (null and conditional) or even multiple modalities in a single stage of fixation as a gene objective has been a persistent objective of those involved in the technique, as well as genetic engineering. The community of end users. In fact, two methods have tried to address this need: FlEx and Inactivation-first (KO-first).

The FlEx method has been used for both objective and objective fixation as a gene trap (GT), but the basic design principles are the same regardless of the final application. A basic design of a FlEx is shown in FIG. 1, representing U, p. eg, a DSC and representing D an informer. The result of the recombinase action in the FlEx construct is the permanent suppression of the U element (e.g., the DSC) and the inversion (and expression) of the D element (e.g., the reporter).

In its original implementation and application, FlEx was developed as a method to treat conditional alleles by genetic engineering. FlEx was used for the first time to generate a "conditional null" allele for Rarg, by inserting the FlEx cassette into this gene, so that a loxP / lox511 cuplet was inserted upstream of Rarg exon 8, and the rest of the FlEx cassette - composed 5 'to 3' of SA-lacZ-SV40poliA (a GT type element) in the antisense orientation with respect to Rarg, and then another loxP / lox511 cuplet in the antisense orientation with respect to the first loxP / cuplet lox511, and which contains a neomycin phosphotransferase (neo) mini-gene in the direction felt within the intron-8 in Rarg. This design reinforces the Cre-mediated inversion of the GT-type element SA-lacZ-SV40poliA so that it it enters the sense chain and acts as a gene trap; simultaneously, Rarg exon 8 is placed in the antisense orientation (effectively guaranteeing that, even in the case where transcription does not end at the end of the type element GT SA-lacZ-SV40poliA, exon 8 no will be incorporated into the reading message), while neo is eliminated simultaneously, and thus results in a null Rarg allele in which Rarg's expression is replaced by lacZ.

However, despite the success of this method in generating a null allele (due to exposure to Cre), Rarg's unreordered FlEx (pre-Cre) allele was not a true conditional null as originally designed, but a severe hypomorphic allele, where the expression of RargFLEx was significantly reduced compared to Rarg. As a result, RargLEx / FLEx mice show a phenotype that resembled a less severe form of inactivated Rarg mice, and revealing the inability of this initial FlEx embodiment to generate a true conditional null allele.

The FlEx method has also been adapted for use in gene trapping. In this variation of the method, a GT element (SA pgeo-polyA, in which pgeo is a fusion in lacZ framework with neo open reading frames, therefore, the combination of the ability to inform through LacZ and select through Neo) was flanked by two series of FlEx type, an external series composed of a FRT / FRT3 cuplet and an internal series composed of a loxP / lox511 cuplet, both in the mirror image configuration with respect to the other. In this way, the successful incorporation of the resulting GT vector into actively transcribed genes results in the expression of pgeo and, therefore, would allow the selection of these events by selecting G418 and, depending on the site of incorporation of the GT element theoretically it also results in the generation of functional null alleles for the corresponding genes. Once a gene has been trapped to generate the corresponding FlEx allele, the resulting allele may be an inactivated allele or a hypomorphic allele, that is, one in which the expression of the trapped gene is regulated downwards. Treatment of these FlEx alleles with FLP recombinase should, in principle, reverse the GT element to the anti-sense chain, thereby relieving the termination of transcription within the trap element and, therefore, converting the modified gene. in conditional GT. This conditional GT, now "hidden" in the antisense thread, can be reactivated by exposure to Cre, which will reverse it by acting on the loxP / lox511 coupons of the FlEx series.

This application of FlEx technology is based on a GT element to generate null alleles. Therefore, it is subject to the limitations of gene trapping technology, which does not guarantee that true inactivation is generated and the additional risk of inactivating regulatory elements is assumed (due to insertional inactivation

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random). Both the disposition of the GT element, and the degree to which it is effective in terminating transcription can affect whether any given allele will be a null allele. An additional problem is that for the majority of genes that do not have an established function, and even more so one that links the gene to a specific phenotype by studying a definitive null allele, it is very difficult to conclusively prove that a GT allele It is truly a null allele. In fact, for these and other reasons, mostly technical, after 4 years of adoption and the use of a consortium of large-scale mouse mutagenesis - EUCOMM - the FIEX-based gene trapping method has been abandoned in favor of the fixation as a gene target using the KO-first method.

Similar to the FlEx method, current typical KO-first alleles rely, at least in part, on a GT-type element (either SA-lacZ-polyA or SA pgeo-polyA) to generate a similar inactivated allele. However, in recognizing the limitations that have been associated with that approach (effectively learning from the experience gained with the GTs, as well as theoretical considerations), the KO-first also requires that the critical exon flanked by loxP sites downstream of the GT type element must be deleted (using Cre) in order to generate a true null allele. Therefore, in practice, this method first requires the placement of a GT-type reporter / GT cassette flanked by a FRT plus a drug mini-gene in an intron of the target gene somewhere upstream of the exon to be suppressed, while that flanking loxP sites simultaneously the exon programmed to be suppressed. This exon has been referred to as the "critical exon," and regardless of the criteria used to define the "critical exon," the KO-first method clearly requires its separation in order to make the resulting allele. Be a true null. Therefore, after the objective is set, the resulting allele is neither a true null allele nor a conditional null allele. The reasons why the resulting allele is not a true null have been attributed to the fact that, without the separation of the critical exon (which is flanked by loxP sites) by Cre, there is still the possibility of the revised transcription and cutting and splice around the GT type cassette, as well as the transcription of the gene message downstream of the GT type cassette due to the presence of the drug mini-gene. The reason that the resulting allele is not a conditional null allele lies in the fact that without the separation of both the reporter cassette / GT type and the drug mini-gene, the generation of the normal message cannot occur (normal composition , as well as the level and sites of expression).

Therefore, depending on the use, the KO-first conditioned null or null alleles must undergo a second post-fixation stage as an objective. In the event that a true null is desired, the KO-first allele must be treated with Cre recombinase to suppress the critical exon (which is flanked by loxP sites).

On the contrary, a conditional allele can be generated after a Flp-mediated separation of the reporter cassette / GT type and the mini-drug gene, which are together flanked by FRT sites. Thus, the only modifications that remain are an FRT site and the "critical" exon flanked by loxP sites. This allele, in turn, can become null by Cre-mediated separation of the exon flanked by loxP sites.

Although the KO-first method addresses some of the limitations of FlEx, it is still hampered by three main drawbacks that limit its usefulness: first, despite rectifying the lack of reliability of Gt-type elements to generate a true KO-first , fails to provide a true KO-first, without a post-fixation stage as an additional objective; secondly, due to the criteria used to define "critical exons", the KO-first is limited to protein coding genes, effectively placing out of reach of all non-protein coding genes (i.e., those encoding RNAs' no encoders', a class of very important biomolecules). In addition, of the protein coding genes only those for which a "critical exon" can be defined are susceptible to a KO-first design. The criteria for defining a critical exon are that its deletion results in a frame shift between the part of the open reading frame (ORF) that precedes it and the part of the ORF that follows it. This is because the induction of this frame shift is mandatory for the KO-first method to provide definitive inactivation. Therefore, even certain classes of genes encoding proteins are not susceptible to a KO-first design. These include genes in which the ORF is contained within an exon, and genes in which all or most of the exons in tandem are left out in the same frame. Third, once the KO-first allele has become a conditional null allele (by the action of Flp), the resulting allele does not provide any mechanism for the affirmative nullity report after the conversion of the conditional allele to null state Typically, inactivated alleles first separate the reporter (e.g., lacZ) together with the DSC using (a step achieved using recombinant Flp, such as SA-lacZ-polyA and DSC used in KO-first are FRTed) leaving behind sf only the exon flanked by loxP sites (plus a FRT site upstream of it). Therefore, there is no option of conventional first-inactivation approaches for an informant function to report the achievement of a null allele.

Multifunctional alleles

A multifunctional allele (MFA) approach is provided that allows the separation or inactivation of a nucleotide sequence in a genome by the introduction of a set of functional elements that comprise a driving sequence (confirming separation or inactivation), resulting a true inactivation, and which also contain one or more conditional elements whose expression in the allele is conditional (that is, it depends on certain molecular or signal events) and resenable.

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The MFA approach provides goal setting options to generate true inactivation-first alleles that do not require a second goal post-fixation stage to convert the target-set alleles into a null state, thus providing an advantage and conceptual advance over alleles of Inactivation-first typical topics that require a target post-fixation stage to convert alleles set as target into null alleles. The MFA approach is also not limited to use with “critical exons” and is not limited to inactivations due to displacement of the frame, but is generally applicable to modify any nucleotide sequence of interest. MFAs provide enhanced versatility and a multiplicity of options of alleles after a single target setting stage.

The MFA approach provides a true KO-first allele that provides the opportunity to create a second state in the genome of the recipient after the inversion of any selected sequence linked to a drive sequence, and where it can be reviewed after transcription by Inversion of the selected sequence. The selected sequence can be, e.g. For example, a COIN, which, without limitation, can itself comprise a drive sequence that, e.g. For example, it comprises a repressor to control the transcription of another gene or regulatory sequence. In one example, a single stage of target fixation by placing an MFA in a locus can allow the modification of a wild-type locus to a particular state, State A (eg, an inactivation of an endogenous gene). The MFA is designed to allow a change from one state to another particular state, State B (eg, the restoration of a wild-type phenotype) through the action of a first recombinase. State B can be converted to State C (eg, restoration of inactivation and expression of a COIN) by a second recombinase, and so on. Therefore, different states can be reached in a selected locus from a single initial allele when MFAs are used.

The MFA approach can be used to arrange an MFA as a gene trap, so that the transgene comprising the MFA obtains the expression of MFA elements using an endogenous transcriptionally active promoter.

The MFA approach can be used in traditional transgenesis applications where the drive sequence comprises a promoter, exon or exons and introns and, optionally, transcription control elements and followed by a DSC (optional, since it is not necessary for a transgenesis based on a traditional pronuclear injection), an NSI (which may be a second drive sequence) in the antisense orientation with respect to the first drive sequence, and a COIN (which may be a third drive sequence) also provided in the antisense orientation with respect to the first drive sequence.

The MFA approach offers a multitude of options for the creation of loci containing null alleles, conditional null alleles, COINs, drive sequences (which may include reporters) and DSCs, and other elements, in a single goal setting stage. Locus target post-fixation manipulations provide options through the use of recombinant units introduced into the MFA-containing locus, with the MFA construction in the single objective setting stage. The number of different recombinases required to exercise the various manipulable options in the post-fixation locus is reduced by the use of different pairs of cognata site specific recombinase recognition sites that are incompatible (e.g., a pair of FRT sites and a pair of FRT3 sites, a pair of loxP sites and a pair of lox2372 sites, etc.). Therefore, exposure of a locus containing MFA to a single recombinase can independently act on at least two different recombinant units, to recombine a unit such that the unit has elements of interest (eg, reporters, DSCs). , exons, COINs, etc.) in desired orientations, as well as to re-orient specific site recombinase recognition sites within the recombinant unit to form new recombinant units.

The MFA approach provides an option for a COIN that may contain any desired sequence, including but not limited to an informant, or a cDNA encoding a mutant or variant form of the target gene or part of the target gene, or relatives and homologs of the gene target, or even non-protein coding sequences such as microRNAs or clusters of microRNAs, or any combinations of these elements (since they can be accommodated by the placement of internal ribosome entry sites or "self-cleavage" peptides - depending on the choice of elements-between different elements).

The MFA approach provides an option where inactivation is achieved by target setting, a first recombinase is used to restore the inactivated element back to an active (wild type) state, and a second recombinase is used to restore the inactivation, where a COIN is arranged in a direction concomitant with the restoration or inactivation of the inactivated element, thus restoring the restoration of inactivation in the cells in which the second recombinase has been activated.

Although current inactivation-first approaches lack a mechanism to report nullity after the conversion of a conditional to null allele, the MFA approach provides an option for an information element (e.g., a COIN, see FIG 5 and FIG. 6, in the lower part; detectable by genotyping and / or by visualization or other qualitative or quantitative determination, optionally at the cell level) which, after the action of a second recombinase inverts or inverts and cleaves a sequence of nucleotides of interest (e.g., an exon and the surrounding sequence, NSI in FIG. 5 and FIG. 6) and arranges the reporting element in a directional direction,

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effectively reviewing the investment and / or excision of the NSI. In this embodiment, a null allele after a single target fixation stage is converted, by a first recombinase, into a restored allele (in this embodiment, NSI of FIG. 5 and FIG. 6 is an exon and surrounding the sequence or the gene or part thereof replaced by the orientation vector), and by a second recombinase in a null allele that reports its presence by means of the disposition of the COIN in the sense orientation.

Therefore, the MFA approach provides an option for the evaluation of a phenotypic effect of an inactivation (after the target setting stage), then exposure to a first recombinase to restore the inactivated exon or gene or region of the same, evaluating the phenotypic effect of the restoration - that is, the conversion back to the wild type (a stage equivalent to a complementary trial, but devoid of the requirement to generate a new line of transgenic mice, a requirement that has traditionally accompanied the analysis of complement), then optionally exposing the second recombinase to restore inactivation and evaluating the phenotypic effect of restoring the null allele. Therefore, the MFA allele combines a true first-inactivation approach with the versatility of additional (conditional) elements, and the ability to perform an analysis of the true complement type in a genetically modified animal, in a protocol comprising a only goal setting stage.

In an application of the MFA, a method for a complement assay is described, which comprises targeting an endogenous allele of a cell with an MFA according to the invention, then, in a post-setting stage of generating a conditional allele. null from the MFA (by exposure to a first recombinase), wherein the nucleotide sequence of interest in the MFA comprises an exon or an exon plus surrounding sequence, or another region of interest (associated, for example, with a phenotype) in the sense orientation, and evaluate the phenotypic effect of the null conditional allele (which should be wild type). In a further embodiment, the MFA is further exposed to a second recombinase that restores the nullity and, optionally, a phenotypic effect is measured again. In a specific embodiment, the second recombinase also provides a conditional reporter (e.g., a COIN) in the sense orientation, where the conditional reporter reports the conversion of the null conditional allele into a null allele or, where appropriate, Report the nullity reset.

In one embodiment, the NSI comprises an exon and a neighboring intronic sequence, or an exon-intron region of a target gene. In another embodiment, the NSI comprises a region that encodes a cRNA, microRNA, microRNA cluster or other small cnc (s).

MFAs are alleles that can be randomly arranged or targeted at a place of choice in a genome. The MFA is treated by genetic engineering to produce null, conditional alleles or a combination of conditional / null alleles, through a judicious arrangement of the sequences between a series of pairs of cognatas site specific recombinase recognition sites. The resulting alleles, produced by the arrangement of constructs in a genome, are manipulable by selected recombinases, which can be introduced into the construction in the genome transiently or through the breeding of an animal comprising the construction in its genome with a genome. animal comprising a gene for a selected recombinase (eg, a strain expressing Cre, Flp or PhiC31 \ int).

In various embodiments, methods and compositions are provided to generate a true first-inactivation allele, in which nullity does not depend on the performance of a second stage such as, e.g. eg, the separation of a "critical exon" or a "critical region", by the action of a recombinase. Accordingly, embodiments are provided to generate an allele that is multifunctional in a single target setting stage, since it is a true first-inactivation allele with an informer, achieved in a single stage of recombination and goal setting .

The methods and compositions for generating alleles inactivated by the MFA approach are not limited by a requirement to generate a frame shift through the deletion of the investment of a critical exon to generate a null allele, as required by some types. of inactivated alleles (e.g., KO-first or some embodiments of FIEx). Instead, the MFA method is based on its ability to separate the NSI from the transcription unit of the target gene at the time of the target setting, while simultaneously replacing the expression of the NSI with that of a sequence of drive The drive sequence may comprise a GT-type element (e.g., an informant such as SA-lacZ-polyA), a cDNA, an exon or exons, regulatory elements (e.g., enhancers, isolators, operators). Since the drive sequence is defined by the experimenter, alleles that are not null can be done equally well. For example, the drive sequence can encode a dominant negative gene or a constitutively active gene or an activated form of a gene.

In various embodiments, the MFA comprises a nucleotide sequence of interest and a COIN that are each in antisense orientation in the resulting allele, and which further comprise an actuation sequence and / or a DSC in both direction oriented in the allele (or in sense and antisense orientations, or each independently in sense or antisense orientation), whereby after exposure to a first recombinase the drive sequence and / or the DSC are suppressed, the nucleotide sequence of interest is It invests in a meaningful orientation and the COIN remains in the antisense orientation. The allele also includes recombination sites located in order to allow subsequent simultaneous inversion by a second recombinase of the

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nucleotide sequence of interest and the COIN, so that after the action of the second recombinase, the nucleotide sequence of interest is arranged in antisense orientation and the COIN is arranged in a sense orientation. In a further embodiment, the nucleotide of interest is suppressed after treatment with the second recombinase, leaving the COIN in the sense orientation. In a specific embodiment, the COIN is an informant or a DSC. In another specific embodiment, the nucleotide of interest is an exon or region of interest of a gene of a species (e.g., mouse exon, rat, non-human primate or human being) and the COIN is an exon of a gene of another species (e.g., mouse exon, rat, non-human primate or human being).

The MFA approach also allows a gene trapping approach. In this embodiment of the MFA approach, an MFA is inserted into a transcriptionally active locus. This can be achieved by random recombination, or by "target entrapment" (see, eg, U.S. Patent No. 7,473,557). An actuation sequence preceded by a splice acceptor and a splice region and followed by a polyA signal provides an inactivation or "demolition" of any existing transcribed genomic sequence. The inclusion of a DSC without promoter, that is, one whose expression depends on the insertion within the sense chain of a transcriptionally active locus, ensures the positive selection of cells containing the MFA. The inclusion of a nucleotide sequence of interest (NSI) in antisense orientation, together with a COIN in antisense orientation, in conjunction with a recommended provision of site-specific recombinase recognition sites, provides the ability to conditionally express NSI (a from the trapped locus promoter), after exposure to a first recombinase. Then, after exposure to a second recombinase, the expression of the NSI can be deactivated and at the same time replaced by that of the COIN. The DSC without promoter will ensure that any selected cell has the ability to express NSI without promoter and COIN without promoter, and that the expression will be made in accordance with the endogenous expression pattern of the transcriptionally active locus.

Certain advantageous approaches using MFAs are conveniently described in relation to particular embodiments (i.e., with reference to alleles comprising specific sites called recombinase and nucleotide sequences as shown in the figures) for convenience and not by way of limitation, i.e. , recombinases and recombinase recognition sites, drive sequences, reporters, DSCs and nucleotide sequences of suitable interest may be routinely chosen based on the description herein. The "nucleotide sequence of interest" or "NSI" can be any nucleotide sequence of interest, e.g. eg, an exon, an exon plus flanking sequence (s), two or more exons, a fragment of a coding sequence, a complete coding sequence, a regulatory element or sequence, a non-protein coding sequence, an intron, or any of its combinations, etc. COINs may comprise cDNAs, as well as non-protein coding sequences and may incorporate elements such as signals and polyadenylation sites, microRNAs or other non-protein coding RNAs, IRES, codon jump peptides, and any combination thereof. Certain COINs and some systems to use them can be found, e.g. e.g., in US Pat. UU. No. 7,205,148.

Methods and compositions are provided to produce and use MFAs in any cell, including non-human animal cells and in non-human animals. The methods and compositions can be employed using homologous recombination (or random integration) to arrange useful alleles at any selected site (or random site) in the genome of a cell. The methods and compositions can be used in pluripotent, induced pluripotent and totipotent cells. Cells suitable for use with the methods and compositions include ES cells, e.g. eg, of mouse, or rat ES cells. In various cases, true KO-first alleles are described that grant an option for conditional functionality with an integrated reporter function.

In FIG. 2 illustrates an example of how an arrangement of recombinase recognition elements and sites can be designed to create a construct that will eliminate the function of the target gene (i.e., create a null allele), or to alter the function of the target gene ( e.g., turning it into a dominant negative allele, constitutively active or hypomorphic), while at the same time integrating all the downstream elements that would allow (a) the generation of a conditional allele, and (b) its inversion to a null With an informant.

FIG. 2 shows an embodiment of an MFA that can be arranged in a genome (eg, shown using homologous arms to the left and right of the MFA). The objective post-fixation, the resulting allele can be converted into a conditional allele, which is achieved by eliminating a first selected sequence and reversing a second selected sequence. Deletion and inversion can be achieved by the same recombinase or a different recombinase. For example, two pairs of incompatible Flp recognition sites can be used - one to direct the deletion and the other to direct the investment. An example of two such Flp sites are FRT sites and FRT3 sites. In another example, two pairs of incompatible Cre sites can be used, e.g. eg, loxP and lox2372- one to direct the deletion and the other to direct the investment. In addition, two different recombinases can be used (eg, a pair of LoxP sites with Cre and a pair of FRT sites with Flp). For this embodiment, any suitable sites can be chosen, provided that the sites can direct the deletion and inversion of pairs of MFA recombinase sites shown in FIG. 2 (specific embodiments shown in FIG. 5 and FIG. 6).

Although the construction design of FIG. 2 can be used with any of the sequences of interest (ie, NSI is any sequence of interest), the construction design can be particularly useful for replacing an exon of interest with a modified exon.

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In one embodiment, the NSI is an exon (or exons) that occurs naturally, and the COIN is a modified exon (eg, an exon that comprises a mutation). MFA is arranged in a genome of, e.g. eg, a mouse ES cell by, e.g. For example, homologous recombination (using suitable mouse homology arms) and the ES cell is used to produce a genetically modified mouse comprising the construction in the germline of the mouse. In one embodiment, the change in state of the exon that occurs naturally to the modified exon is achieved by the action of a recombinase in the MFA.

In one embodiment, the construct is arranged in a genome of, e.g. eg, a mouse, and the mouse also comprises a recombinase (eg, Cre) whose activity can be regulated. A recombinase can be regulated, e.g. for example, using a fusion protein that disposes of the recombinase under the control of an effector or metabolite (eg, CreER, whose activity is positively controlled by tamoxifen), arranging the recombinase under the control of a tissue specific promoter , or by arranging the recombinase under the control of a promoter (or other regulatory element) that is active at a particular stage of development (e.g., a Nanog promoter), or an inducible promoter (e.g., one whose activity is controlled by doxycycline and TetR or variants of TetR), or combinations of these technologies.

The MFA embodiment shown in FIG. 2 carries elements comprising a sequence encoding a drive sequence, a DSC, a nucleotide sequence of interest (NSI) and a COIN, wherein the elements are arranged between a series of recombinase recognition sites that are selected with the in order to provide a desired functionality to the MFA.

The upper part of FIG. 2 illustrates a nucleotide sequence of interest (NSI) in a genome of choice (eg, an NSI in a mouse genome). An MFA as shown is introduced into the genome, e.g. eg, by homologous recombination to replace the NSI. The NSI is replaced by the MFA shown, in which the NSI of the MFA is reversed as shown and, therefore, is no longer incorporated into the transcription of the target gene. The presence of the MFA can be conveniently confirmed if the drive sequence contains a reporter (eg, a lacZ). A DSC is also present, to assist in the selection of modified cells (eg, mouse ES cells modified with the MFA).

The MFA of the embodiment of FIG. 2 comprises five distinct units of the sequence, which are defined by five sets of recombinase recognition sites. FIG. 3 contains a conceptual representation of the five different sequence units flanked by compatible recombinase recognition sites.

The first distinct recombinant unit comprises R1 / R1 'sites (e.g., FRT3 sites) in opposite orientation (i.e., directing an inversion), where between the R1 / R1' sites the following is arranged: a drive sequence (a 3 'region of splicing and acceptor 5' with respect to the drive sequence, and a 3 'polyA signal with respect to the drive sequence, are not shown in FIG. 3 for the sake of simplicity), an R2 site (e.g., a Rox site), a DSC, an R3 site (e.g., a FRT site) in the same orientation as the R1 site, an R4 site (e.g., a loxP site) and a nucleotide sequence of interest (NSI) in antisense orientation with respect to the direction of transcription (ie, encoded by the antisense chain) of the target gene, and an R5 site (e.g., a site lox2372) in the same orientation as the r4 site. In cases where an R3 'site (in opposite orientation of the R3 site shown in FIG. 3A) is further included downstream of the 3' R1 'site, the unit in the presence of a recombinase that recognizes R1 / R1' will reverse the NSI to a position for transcription and will suppress the drive sequence and the DSC. In one embodiment, an additional sequence includes a COIN disposed in the antisense chain and is followed by an R4 'site (e.g., a loxP site) that is in opposite orientation with respect to the R4 site of the unit, so that After exposure to a recombinase that recognizes R4 / R4 '(e.g., Cre), the COIN is reversed so that the coding sequence of the COIN is now in position for transcription downstream of the NSI.

The second distinct recombinant unit (FIG. 3B) comprises R2 / R2 'sites (e.g., Rox sites) in the same orientation (i.e., directing a deletion), which comprises the following sequences arranged between the R2 / R2 sites ': a DSC, a first R3 site (e.g., a first FRT site) in the same orientation as the R1 site of the first different recombinant unit, a first R4 site (e.g., a first loxP site) , an NSI in inverted orientation (ie antisense) with respect to the target gene, a first R5 site (e.g., a first lox2372 site) in the same orientation with respect to the R4 site, an R1 'site (p. eg, a second FRT3 site) and an R3 'site (for example, a second FRT site), both in opposite orientation to the R3 site, a COIN (in antisense orientation with respect to transcription of the target gene), an R5 site '(e.g., a second lox2372 site) in the same orientation as the R5 site, and an R4 site' (e.g., a second loxP site) in the same orientation as R4 . This second distinct recombinant unit is cleavable by a recombinase that recognizes R2 / R2 '. When included in the MFA, this unit can be split to leave behind a drive sequence (e.g., in some embodiments, an informant, e.g., a sequence encoding lacZ), flanked by a site R1 and a R2 or R2 'site.

The third distinct recombinant unit (FIG. 3C) comprises R3 / R3 'sites (e.g., FRT sites) in opposite orientation (ie, directing an inversion), which comprises the following sequences arranged between the R3 / R3' sites : an R4 site (e.g., a loxP site), an NSI in reverse orientation (i.e. antisense) with respect to transcription of the target gene, an R5 site (e.g., a lox2372 site) in the same orientation as the site R4 of the unit (ie, of FIG. 3C) and a site R1 '(e.g., a FRT site) in the orientation opposite to the site R3. This unit can be reversed by

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the action of a recombinase that recognizes R3 / R3 '(e.g., a Flp recombinase, in which the R3 / R3' sites are FRT sites), resulting in the arrangement of the NSI in the proper orientation for transcription And the translation.

The fourth distinct recombinant unit (FIG. 3D) comprises R4 / R4 'sites (eg, two loxP sites) in the same orientation (ie, directing a deletion), which comprises the following sequences arranged between the R4 / sites. R4 ': an NSI in reverse orientation (i.e. antisense), a site R5 (e.g., a first site lox2372) and a site R1' (e.g., a site FRT3) and a site R3 '( e.g., a FRT site) each in the same orientation with respect to the R4 site, a COIN (in antisense orientation) and an R5 'site (e.g., a second site lox2372) in the same orientation as the site R5. In the presence of a recombinase that recognizes R4 / R4 '(e.g., Cre if R4 / R4' are loxP sites, e.g.), this unit is cleavable. If available within the MFA and exposed to recombinant R4 / R4 '(in the absence of exposure to a recombinase that recognizes R1 / R1', R3 / R3 '), this unit will be suppressed and leave the drive sequence (p. eg, in some embodiments an informant, eg, a sequence encoding lacZ) and the DSC. Therefore, this unit allows an embodiment in which the MFA, when replacing a sequence in a genome (e.g., replacing an exon), can act in the presence of a recombinase that recognizes R4 / R4 'as an allele null comprising a drive sequence and a DSC. The DSC of the MFA can be separated, if desired, after the action of a recombinase that recognizes R2 / R2 '(e.g., a Dre recombinase in which R2 / R2' are Rox sites) because the DSC is flanked upstream and downstream through sites R2 / R2 'in the same orientation.

The fifth distinct recombinant unit (FIG. 3E) comprises R5 / R5 'sites (e.g., two lox2372 sites) in the same orientation (ie, directing a deletion), as well as in the same orientation of the R4 / sites. R4 'of the fourth distinct recombinant unit, comprising the following sequences arranged between sites R5 and R5': a site R1 '(e.g., a FRT3 site) and a site R3' (e.g., a site FRT) in the same orientation with respect to each other, but in orientation opposite to the R1 site of the first different recombinant unit, and a COIN (in antisense orientation) with respect to transcription of the target gene.

As those skilled in the art will recognize, overlapping recombinant units are described in this way to transmit the structure of the MFA, rather than limiting the possible recombinant elements in the MFA. For example, those skilled in the art will recognize that each of the recombinant units comprises site-specific recombination sites within the recombinant unit, and that the action of a recombinase at the sites (through recombinant units) achieves desired manipulations and MFA described that achieve expected functions of the MFA. For example, with reference to FIG. 3, the action of a recombinase that recognizes R1 / R1 'and R3 / R3' functions to manipulate portions of all five recombinant units as conceptually shown in FIG. 3.

Once an MFA is arranged in a desired place in a genome, it can be treated by genetic engineering in such a way that it provides a null allele with an informative function, where the null allele can be modified again (in the post-fixation stage target, recombinase-mediated stage) so that it lacks all sequences flanked with recombinase recognition sites oriented in the same direction. An example of this embodiment is shown in FIG. 4, which shows examples of suitable recombinase recognition sites, where all elements other than the drive sequence (here, encoding lacZ) are flanked upstream and downstream by Rox sites. After exposure to Dre recombinase, only the drive sequence is present. The cleavage of the sequences with Rox sites can be confirmed by the loss of the DSC (here, containing neor), and / or the loss of the COIN and / or the loss of the NSI. The result is a true null allele that lacks DSC, NSI and COIN.

An MFA can be used as illustrated in FIG. 2 and as exemplified in the upper part of FIG. 3 to create a conditional allele. A conditional allele can be generated by selecting the appropriate recombinase with which the allele is exposed in the first instance. The appropriate recombinase in this embodiment is a recombinase that reverses the NSI back to the sense chain and leaves the COIN in the antisense orientation. This can be achieved, for example, by exposing the MFA to a recombinase that recognizes R1 / R1 'and also R3 / R3' (e.g., a Flp recombinase, wherein R1 / R1 'and R3 / R3' are select from FRT and FRT3 sites; see Fig. 5 for a particular embodiment). Simply put, once an MFA is arranged at a desired location in a genome, it can be used to generate a conditional allele, where the inverted NSI of FIG. 2 and the top of FIG. 3 are arranged in an orientation for transcription of the target gene, while leaving the COIN in antisense orientation and the drive sequence and the DSC are suppressed. An example of this embodiment is shown in FIG. 5, in which a drive sequence containing a lacZ and a DSC containing neor are separated by first exposing the allele to the Flp recombinase, causing an inversion of elements directed by FRT3 sites, followed by the Flp-mediated deletion directed by FRT sites . The resulting allele presents the NSI in a transcription orientation, but leaves the COIN in the antisense orientation.

As shown in FIG. 6, the same conditional allele can be achieved if Flp-mediated investment occurs first through FRT sites (as in FIG. 5) or FRT3 sites (as in FIG. 6).

In the embodiment that generates a conditional allele, recombinase sites that remain in the allele are selected so that treatment with one or more suitable recombinases results in the subsequent deletion of the NSI (or re-inversion of the NSI) and the inversion of the COIN, so that the allele results in a null allele with respect to the NSI, but also places the COIN in the orientation for transcription. An example of this is shown using sites

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loxP and lox2372, each of which independently directs Cre-mediated recombination. Although Cre-reactive sites are used, any suitable sites can be used instead of the Cre sites.

As shown in FIG. 7, the NSI in sense orientation (that is, in the position for transcription and translation) is arranged 3 'with respect to a first lox2372 site. After the NSI, a first loxP site is found in the same orientation as the first lox2372 site, and an inverted (i.e. antisense) COIN is disposed downstream of the first loxP site, and the inverted COIN disposed upstream of a second site lox2372 in opposite orientation with respect to the first site lox2372. Arranged downstream of the second lox2372 site is a second loxP site arranged in an opposite orientation with respect to the first loxP site. This provision allows, after treatment with Cre, the inversion through any of the lox sites followed by its deletion through any of the lox sites (see FIG. 7). The resulting allele contains a COIN in sense orientation, that is, in the position for transcription and translation.

In an alternative arrangement (see FIG. 8), a loxP site is arranged 5 'with respect to the NSI (instead of arranged between the NSI and the COIN), so that exposure to Cre results in the inversion of the NSI to the antisense orientation and the COIN to the sense orientation.

The MFA approach provides options for many accomplishments. In a specific embodiment, after exposure to the first recombinase, the arrangement of recombinase elements and sites are as shown in the construction of the bottom part of FIG. 5 or FIG. 6, where the FRT3 site as shown is the R1 site that does not have a cognate site in the resulting allele, the FRT site as shown is an R3 recombinase site that does not have a cognate site in the resulting allele, The lox2372 site on the left is the R5 site that matches a R5 'site of recombinase cognate that occupies the lox2372 site on the right as shown, the loxP site on the left as shown is the site R4 that pairs with an R4 'site of recombinant kinase provided by the loxP site on the right as shown, and the Rox site as shown is the site R2 that does not have a recombinant site of kinase in the resulting allele .

In a specific embodiment, after exposure to the second recombinase, the arrangement of recombinase elements and sites of the resulting allele is as shown in the construction of the lower part of FIG. 7, where the FRT3 site shown is the R1 site that does not have a cognate site in the resulting allele, the lox2372 site is the R5 site that is not paired with a recombinant cogninate site in the resulting allele, the FRT site shown is the R3 site that is not paired with a recombinant kinase site in the resulting allele, the loxP site as shown is the R4 site that is not paired with a recombinant kinase site in the resulting allele, and the Rox site as Sample is the R2 'site that is not paired with a recombinant site of cognate in the resulting allele.

In a specific embodiment, the resulting allele allows COIN expression after exposure to the second recombinase. In a specific embodiment, the COIN is an informant or a DSC.

An MFA is described comprising a COIN, an NSI, a DSC, a reporter and recombinase sites that are arranged so that the action by a recombinase will cleave the COIN, NSI and DSC, but not the reporter (FIG. 11 B), while the action with a different recombinase will generate an allele that lacks the DSC, but that has the NSI in a direction oriented, while maintaining the COIN in antisense orientation (FIG. 11C). This resulting allele has recombinase sites arranged so that the action such by an additional recombinase will cleave the NSI and arrange the COIN in a sense orientation (FIG. 11D). Therefore, in the aforementioned embodiments, this MFA will allow the selection of a true inactivation with an informer function and the separation of the DSC, or the placement of an NSI, where the subsequent separation of the NSI is confirmed by the provision concomitant of a COIN in direction orientation. A schematic diagram of some overlapping recombinase units is shown in FIG. 11A for such an allele, with the recombinase units represented by dashed shapes.

An MFA is described which comprises a COIN, an NSI, a DSC, an informant and recombinase sites that are arranged so that the action by a recombinase will cleave the NSI and the DSC, but will maintain the orientation of the informant and the COIN (Fig. .12B), while the action with a different recombinase will generate an allele that lacks the DSC and the informer, but that has the NSI in the direction oriented, while maintaining the COIN in antisense orientation (Fig. 12C). This resulting allele has recombinase sites arranged so that the action by an additional recombinase will cleave the NSI and arrange the COIN in a sense orientation (FIG. 12D). Therefore, in the aforementioned embodiments, this MFA will allow the selection of a true inactivation with an informer function and the separation of the DSC; or the provision of an NSI, where the subsequent separation of the NSI is confirmed by the concomitant provision of a COIN in a meaningful orientation. A schematic diagram of some overlapping recombinase units is shown in FIG. 12A for such an allele, with the recombinase units represented by dashed shapes.

An MFA is described which comprises a COIN, an NSI, a DSC, a reporter and recombinase sites that are arranged so that the action by a recombinase will cleave the reporter and the DSC and arrange the NSI in a sense orientation (FIG. 13B) . This resulting allele has recombinase sites arranged so that the action by an additional recombinase will arrange the NSI in antisense orientation, while arranging the COIN in

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direction orientation (FIG. 13C). Therefore, in the aforementioned embodiments, this MFA will allow the creation of a conditional allele from an MFA.

An MFA is described which comprises a COIN, an NSI, a DSC, a reporter and a different series of recombinase sites that are arranged so that the action by a selected recombinase will cleave the reporter and the DSC and arrange the NSI in a meaningful direction. (FIG. 14B). This resulting allele has recombinase sites arranged so that the action by an additional recombinase will arrange the NSI in antisense orientation, while arranging the COIN in a sense orientation (FIG. 14C). Therefore, this MFA will allow the creation of a conditional allele from an MFA.

An MFA is described comprising an NSI, a DSC, a reporter, a COIN and recombinase sites that are arranged so that the action by a selected recombinase will cleave the reporter and the DSC and arrange the NSI in a meaningful orientation, while keep the COIN in antisense orientation (FIG. 15B). This resulting allele has recombinase sites arranged so that the action by an additional recombinase will arrange the NSI in antisense orientation, while arranging the COIN in a sense orientation (FIG. 15C). Therefore, this MFA will also allow the creation of a conditional allele from an MFA.

EXAMPLES

Example 1: MFA Hprtl

Hprtl is a gene that is linked to X in mice, and ES Hprtl-null cells are resistant to the nucleobase analogue 6- thioguanine (6-TG). This property provides an easy and robust phenotypic test, since cells that are wild-type for Hprtl die in the presence of 6-TG, while cells that are null for Hprtl survive. Additionally, if ES cells that are derived from male blastocysts are targeted (as is typically the case, and is also the case for most ES cell lines currently in use for target setting), then only one is needed. target setting round to generate ES HPRT1MFA / Y cells. In order to generate ES HPRT1MFA / Y cells, an MFA in a target fixation vector according to the allele shown in FIG. 5 (upper part), is prepared by standard genetic engineering methodology and bacterial homologous recombination according to the VELOCIGENE® method described in US Pat. UU. No. 6,586,251 and in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21 (6): 652-659. The Hprt1MFA allele is designed around exon 3, which defines exon 3 and the intronic conserved sequence directly 5 'and 3' thereof as the NSI (FIG. 9). The reason for this choice is that exon 3 begins in frame 2 (f2) and ends in frame 0 (Z0); by extension, the preceding exon (i.e., exon 2) ends in frame 2 (f2) and the next exon (i.e., exon 4) begins in frame 0 (f0). This means that if this NSI is reversed in the antisense orientation, then exon 2 moves outside the frame with respect to exon 4, because exon 2 ends at f2 and exon 4 starts at f0. Thus, if in the Hprt1MFA allele there is some transcription beyond the drive sequence - SA-lacZ-polyA (FIG. 10) - and there is also a splice that separates the drive sequence from the final mRNA, that mRNA will not understand exon 3 and encode a nonsense sequence, effectively leading to an mRNA and Hprt1-null phenotype. On the contrary, for the Hprt1C0IN-INV allele (generated by the treatment of Hprt1MFA with FLP or FLP variants to first generate the Hprt1C0IN allele, then, by treatment with Cre, to generate Hprt1Ca -INV) if there is a transcript after of SA-eGFP-polyA of the COIN element (FIG. 10), and there is also a splice that separates the SA-eGFP-polyA sequence from the final mRNA, that mRNA will not comprise exon 3 and, therefore, will encode a nonsense sequence, effectively leading to an mRNA and Hprt1-null phenotype.

The antisense oriented NSI is exon 3 and surrounds the evolutionarily conserved intronic sequence of Hprt1 (FIG. 9), and the antisense oriented COIN is an SA-eGFP-polyA. The target setting vector has a mouse homology arm upstream of the first FRT3 site and downstream of the second Rox site that direct the target setting at the Hprt1 locus so that it is replaced by its MFA version, so ( a) an SA-LacZ-polyA element in the direction oriented with respect to the direction of the transcription of Hprt1, followed by a DSC in the direction antisense with respect to the direction of the transcription of Hprt1, both exon 3 of Hprt1 above , (b) exon 3 is provided in the antisense orientation with respect to the direction of the transcription of Hprt1, and (c) a COIN element is disposed in the antisense orientation with respect to the direction of the transcription of Hprt1 downstream of the exon 3, and in that these different elements are flanked by site specific recombinase recognition sites, arranged together in recombinant units as detailed in FIG. 3 and FIG. 10, SA-lacZ-polyA being the drive sequence, and NSI being exon 3 plus flanking intronic sequences of Hprt1.

The target fixation vector is prepared and electroporated in ES cells according to the VELOCIGENE® method described in US Pat. UU. No. 6,586,251 and in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21 (6): 652-659. The resulting ES cells carry the MFA allele of Hprt1 instead of the wild type version of Hprt1. Before any further modification, the ES Hprt1MFA / Y cells are resistant to treatment with 6-TG (because they are effectively null for Hprt1), demonstrating the usefulness of the MFA method for generating a true first inactivation allele. After treatment with Dre, this property is preserved, while the genotype of the cells becomes Hprt1SA ~ LacZ ~ polyA / Y. Although for the Hprt1 locus this modification cannot alter even the

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Reporter expression level (LacZ) does not have any phenotypic consequences (alters resistance to 6-TG), this may not be the case for other loci. After treatment with FLP or FLP variants, at a stage that is effectively equivalent to a complementary assay, ES Hprt1MFA / Y cells become ES Hprt1COIN / Y cells that are effectively wild-type and therefore sensitive to 6-TG. In addition, this operation restores the expression of the Hprtl message back to its wild type identity. After treatment with Cre, ES Hprt1COIN / Y cells become ES Hprt1COIN-NV / Y cells that are effectively null for Hprtl and, therefore, resistant to 6-TG. In addition, this operation results in the repeal of the wild-type message expression of the Hprt1 message, and its concomitant replacement by a hybrid message composed of the first exon of Hprt1 and eGFP (encoded by the COIN element), thereby generating an allele that expresses eGFP instead of Hprt1. This new property, the expression of eGFP, can optionally be used to rate the investment of the COIN element to the chain with meaning and also has utility to allow the isolation of cells in which this event has taken place from a population of cells, where both types of cells exist (ES Hprt1COIN / Y cells and ES Hprt1COIN-INV / Y cells). Therefore, not only does the COIN allele become null, but the event is also marked by a new, easily measurable and useful event.

Example 2: Results of the MFA Hprtl

An MFA was constructed that has a lacZ (SA (adml) -gtx-lacZ-pA) reporter in a sense orientation, a neomycin DSC (Neo), an NSI in antisense orientation that encompasses a critical exon (ec) for Hprtl (exon 3) and evolutionarily conserved flanking intronic sequences, and a COIN (Gtx-SA-HA-myc3-TM-T2A-GFP-pA) with a recombinase site arrangement as shown in FIG. 16A. The MFA was electroporalized in ES FlH4 cells and selected for resistance to G418. Subsequently, G418 resistant colonies were genotyped to determine target fixation. Five target clones (Hprt1MFA / Y) were obtained from a total of 96 screened colonies. It was found that all five of these clones survived and propagated when grown in a standard ES cell medium supplemented with 6-TG 10 pM (which is the standard 6-TG survival assay used), as expected for Hprf1-null cells (Doetschman, T. et al. (1987) Targeted correction of mutant HPRT gene in mouse embryonic stem cells, Nature 330: 576-578). In contrast, the parental cell line, F1H4, as well as any of the clones not set as the target that were tested, failed to grow in the presence of 6-TG. These results are in accordance with what has been previously reported (Doetschman et al. (1987)).

After treatment with FLPo recombinase (Raymond, CS. And Soriano, P. (2007) High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells, PLoS ONE 2: e162), the Hprt1MFA allele becomes the Hprt1C0IN allele ( FIG. 16B), giving rise to ES cells Hprt1MFA / Y. This operation results in the separation of the LacZ reporter, the DSC, as well as the restoration of the NSI re-investment in the felt chain. Therefore, the resulting allele (Hprt1C0IN) is functionally wild-type, since the wild-type Hprt1 mRNA is encoded and expressed.

After further treatment with Cre recombinase (Sauer, B. and Henderson, N. (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1, Proc. Natl. Acad. Sci. USA 85: 5166-5170) , the Hprt1COIN allele becomes the Hprt1CONI-NV allele (FIG. 16C), resulting in ES Hprt1C0IN-INV / Y cells. This allele (Hprt1C0IN'INV) is functionally null, since the Hprt1 mRNA is replaced by one that encodes eGFP (and also lacks NSI- that is, exon 3 of Hprt1 and flanking intronic sequences as defined in the design phase).

Cells carrying the MFA (Hprt1MFA / Y) were tested for resistance to nucleotide analogue 6-Tg, and compared with wild-type cells (FIG. 17). ES Hprt1MFA / Y cells survived, while Es Hprt1 + / Y cells died, indicating that Hprt1MFA / Y are functionally Hprt1-null. ES Hprt1MFA / Y cells were then treated with FLPo, to test whether the Hprt1MFA allele becomes the Hprt1C0IN allele. The resulting ES Hprt1COIN / Y cells are expected to be phenotypically wild-type, since the expression of Hprt1 is restored. This has proven to be the case, since ES Hprt1COIN / Y cells die when grown in the presence of 6-TG, as well as their wild type counterparts (Hprt1 + / Y). Finally, the ES Hprt1COIN / Y cells were treated with Cre to generate ES Hprt1COIN-INV / Y cells that are predicted to be null for Hprt1 since the COIN module is activated while simultaneously exon 3 of Hprt1 is suppressed ( FIG. 16, Panel C). When grown in the presence of 6-Tg, ES Hprt1COIN-INV / Y cells survived and proliferated, confirming that they are functionally null for Hprt1, as intended by the design and application of MFA.

The phenotypic results obtained above were further confirmed at the level of proteins, by means of Western transfers of ES cell protein preparations belonging to each of the genotypic classes: (Hprt1 + / Y) wild type, Hprt1MFA / Y (MFA ), Hprt1C0IN / Y (MFA + FLPo) and Hprt1C0IN ~ INV / Y (MFA + FLPo + Cre). These protein preparations were examined for the informant and the expression of NSI (ie, Hprt1). Cells are Hprt1MFA / Y lack the Hprt1 protein, but express the reporter (LacZ). In ES Hprt1C0IN / Y cells, the expression of Hprt1 is restored to wild-type levels, which reflects the disposition of the NSI (exon 3 of Hprt1) again in the sense orientation, and did not show an informative protein (LacZ), which confirms the splitting of the informant by FLPo. This established that the Hprt1MFA allele is in fact null, and can become a functional wild type allele after the separation of the informant and the DSC, and the re-

concomitant investment of the NSI in the felt chain (an operation experimentally performed by FLPo). The fact that at the level of expression of the Hprt1 protein the Hprt1COIN allele is identical to that of the wild type (Hprt1 +), further demonstrates the robustness of this method to generate a true null conditional and perform the equivalent of a complementary test in a post-fixation stage mediated by 5 recombinase. Finally, ES Hprt1CO N ~ INV / Y cells lack the Hprtl protein, which effectively confirms the phenotypic observations made using the 6-TG resistance test. This further confirms that the COIN-based null conditional allele (Hprt1C0IN) works as intended.

Claims (14)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 1. A nucleic acid construct, comprising: (a) target fixing arms to direct the construction of nucleic acid to a target gene of a nucleic acid of a cell; (b) a sense-oriented drive sequence with respect to transcription of the target gene, and a drug selection cassette (DSC) in sense or antisense orientation; (c) in antisense orientation, a nucleotide sequence of interest (NSI) and a COIN (conditional element by inversion); Y (d) recombinant units, wherein the recombinant units comprise two first pairs of cogninate recombinase recognition sites recognized by a first recombinase, a first recombinant unit comprising the driving sequence, the DSC and the NSI, wherein the first recombinant unit is flanked by a pair of the first two pairs of recombinant kinase recognition sites recognized by the first recombinase, a second recombinant unit comprising the NSI, wherein the second recombinant unit is flanked by a pair of the first two pairs of recombinant kinase recognition sites recognized by the first recombinase, two second pairs of recombinant kinase recognition sites recognized by a second recombinase, and a third pair of recombinase recognition sites cognatos recognized by a third recombinase; wherein the first pairs, the second pairs and the third pairs of recombinase recognition sites are recognized by different recombinases; wherein the recombinant units recombine after exposure to a first recombinase to form a conditional allele that lacks the drive sequence and the DSC and contains the NSI in a sense orientation and the COIN in antisense orientation; Y wherein the conditional allele, when additionally exposed to the second recombinase, recombines to form an allele that lacks NSI and that has the COIN in a sense orientation. 2. A method for modifying a non-human cell, comprising: target a nucleotide sequence of interest in a cell with a nucleic acid construct of claim 1 to form a target cell, expose the target cell to the first recombinase to form the conditional allele and expose the conditional allele to a second recombinase that cleaves the NSI and places the COIN in a sense orientation. 3. The method of claim 2, wherein the NSI in the cell is an exon or a nucleotide sequence that is associated with a phenotype. 4. The method of claim 3, wherein the nucleotide sequence of interest in the cell is replaced by the nucleic acid construct of claim 1. 5. The method of claim 4, wherein the phenotype is determined for the first time after forming the target cell, but before exposure to the first recombinase. 6. The method of claim 5, wherein the phenotype is determined a second time after exposure to the first recombinase. 7. A nucleic acid construct, comprising: a drug selection cassette (DSC), a reporter, a COIN, a nucleotide sequence of interest (NSI) and five pairs of recombinase sites arranged between the reporter, the DSC, the COIN and the NSI; wherein no pair of recombinase sites is identical to any other pair, and where a first two pairs of recombinase sites are recognized by the same first recombinase, a second two pairs of recombinase sites are recognized by the same second recombinase and a fifth pair of recombinase sites is recognized by a third recombinase; where the first, second and third recombinases are not equal, and where with respect to the direction of transcription, the informant is in a sense orientation, the NSI is in antisense orientation, the COIN is in antisense orientation and the DSC is in sense or antisense orientation; wherein the recombinase sites are arranged in recombinant units comprising (i) a first recombinant unit comprising the drive sequence, the DSC and the NSI, wherein the first recombinant unit is flanked by a pair of the first two pairs of recombinase recognition sites 5 10 fifteen twenty 25 30 cognatos recognized by the first recombinase; and (ii) a second recombinant unit comprising the NSI, wherein the second recombinant unit is flanked by a pair of the first two pairs of cogninase recombinase recognition sites recognized by the first recombinase; Y wherein the first two pairs and the second two pairs of recombinase sites are arranged so that upon exposure to the first recombinase, a modified allele is formed, wherein the first two pairs of recombinase sites direct the cleavage of the reporter, the cleavage of the DSC, the inversion of the NSI to sense orientation and the COIN is maintained in antisense orientation, and (a) the second two pairs of recombinase sites are arranged so that, after exposure to the second recombinase, the NSI is split and the COIN is placed in a directional direction; or (b) the second two pairs of recombinase sites are arranged such that after exposure to the second recombinase, the NSI is placed in antisense orientation and the COIN is placed in a directional direction. 8. A nucleic acid construct according to claim 7, wherein the fifth pair of recombinase sites are arranged such that, after exposure to the third recombinase and in the absence of exposure to the first or second recombinase, the fifth recombinase cleaves COIN, NSI and DSC, and keeps the informant in the right direction. 9. A nucleic acid construct according to claim 7, wherein the nucleic acid construct is inserted into a gene, and with respect to the transcription direction of the gene, from 5 'to 3', the construct comprises an informant in sense orientation, a DSC in sense or antisense orientation, an NSI in antisense orientation and a COIN in antisense orientation. 10. A nucleic acid construct according to claim 9, wherein the DSC is in a meaningful orientation. 11. A nucleic acid construct according to claim 7, wherein the nucleic acid construct is inserted into a gene, and with respect to the transcription direction of the gene, from 5 'to 3', the construct comprises a COIN in antisense orientation, an NSI in antisense orientation, a DSC in sense or antisense orientation, and a reporter in sense orientation. 12. A nucleic acid construct according to claim 11, wherein the DSC is in a meaningful orientation. 13. A nucleic acid construct according to claim 7, wherein the nucleic acid construct is inserted into a gene, and with respect to the transcription direction of the gene, 5 'to 3', the construct comprises an NSI in antisense orientation, a DSC in sense or antisense orientation, an informant in sense orientation and a COIN in antisense orientation. 14. A nucleic acid construct according to claim 13, wherein the DSC is in a meaningful orientation.